Par1 and par2 c-tail peptides and peptide mimetics

ABSTRACT

The present invention concerns isolated PAR1 cytoplasmic tail (c-tail) peptides and isolated PAR2 cytoplasmic tail (c-tail) peptides, as well as compositions comprising these peptides, uses thereof and methods of treating various diseases, in particular cancer.

FIELD OF THE INVENTION

This invention relates to compositions and methods for treating variouspathologies, in particular cancer, wherein the compositions comprisepeptides and peptide mimetics which inhibit PAR signal transduction.

BACKGROUND OF THE INVENTION

The family of mammalian protease-activated receptors (PARs), belongingto G protein-coupled receptor (GPCR) is composed of four genes. PAR1 isactivated following the release of N-terminal peptide and the exposureof an otherwise hindered ligand. This exclusive mode of activationserves as a general paradigm for the entire PAR family. Expression ofhPar1 and epithelial tumor progression are directly correlated in bothclinically obtained biopsy specimens and a wide spectrum ofdifferentially metastatic cell lines (Even-Ram S, et al. (1998) Nat Med4: 909-914). PAR1 has been shown to play a central role in the invasiveand metastatic cancers of breast, ovaries, lung, colon, prostate andmelanoma (Grisaru-Granovsky S, et al. (2005) Int J Cancer 113: 372-378;Nierodzik M L, et al (1998) Blood 92(10):3694-700; Salah Z, et al (2005)FASEB J 19(1):62-72; Granovsky-Grisaru S, et al (2006) Gynecol Oncol103(3):802-6; Agarwal A, et al (2008) Mol Cancer Ther 7(9):2746-57).

Importantly, PAR1 cellular trafficking and signal termination appear tooccur in a different mode than other GPCRs. Instead of recycling back tothe cell surface after ligand stimulation, activated PAR1 is sorted tothe lysosomes and degraded. Aberrant PAR1 trafficking, resulting inreceptor-populated cell surfaces and causing prolonged and persistentsignals, has been found in breast cancer (Booden M A, et al (2004) MolCell Biol. 24:1990-1999). While cellular trafficking of PAR1 impinges onthe extent and mode of signaling, identification of individual PAR1signaling partners and their contribution to breast cancer progressionremain yet to be elucidated.

Surprisingly, PAR2, the second member which is not considered athrombin-receptor (in contrast to PAR1, 3 and 4) was found to beconnected to coagulation by virtue of its activation by othercoagulation proteases such as tissue factor; TF bound to FVIIa (factorVIIa); TF-FVIIa. PAR2 was associated with promotion of breast cancer (SuS, et al (2009) Oncogene 28(34):3047-57).

SUMMARY OF THE INVENTION

The present invention is based on the identification of a bindinginteraction between a cytoplasmic portion of PAR1 or PAR2 and PH-domaincontaining proteins.

As a non-limiting example, a binding interaction was identified betweenthe cytoplasmic portion of PAR1 or PAR2 and the PH-domain containingproteins Etk/Bmx, Akt and Vav3. Specifically, a PAR1 cytoplasmic-tail(c-tail) peptide was shown to penetrate into cells in culture and tointerfere in the binding reaction between PAR1 and Etk/Bmx.

Moreover, abrogation of the binding interaction between PAR1 and thePH-domain containing protein Etk/Bmx resulted in reduction in PAR1induced oncogenic activity.

Accordingly, by a first of its aspects, the present invention providesan isolated PAR1 cytoplasmic tail (c-tail) peptide selected from thegroup consisting of:

-   -   (a) an isolated PAR1 c-tail peptide capable of interfering in        the binding reaction between PAR1 and a PH-domain containing        protein;    -   (b) an isolated PAR1 c-tail peptide comprising SEQ ID NO: 1;    -   (c) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSIL (SEQ ID NO: 3);    -   (d) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSILCCK (SEQ ID NO: 4); and    -   (e) any fragment or modification of the peptides of (a) (b) (c)        or (d).

In certain embodiments the PH-domain containing protein is selected fromthe group consisting of Etk/Bmx, Akt and Vav3. In a specific embodiment,the PH-domain containing protein is Etk/Bmx.

In another aspect, the present invention provides an isolated PAR2c-tail peptide selected from the group consisting of:

-   -   (a) an isolated PAR2 c-tail peptide capable of interfering in        the binding reaction between PAR2 and a PH-domain containing        protein;    -   (b) an isolated PAR2 c-tail peptide comprising SEQ ID NO: 2; and    -   (c) any fragment or modification of the peptides of (a) or (b).

In another aspect, the present invention provides a method of inhibitingPAR1 mediated signal transduction comprising administering an agentcapable of selectively inhibiting the binding of PAR1 and a PH-domaincontaining protein, in particular Etk/Bmx.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount ofan agent capable of selectively inhibiting the binding of PAR1 and aPH-domain containing protein, in particular Etk/Bmx, or a pharmaceuticalcomposition comprising said agent to a patient in need thereof.

In certain embodiments said agent is a peptide.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount of apeptide, or a pharmaceutical composition comprising the peptide to apatient in need thereof, wherein the peptide is selected from the groupconsisting of:

-   -   (a) An isolated PAR1 c-tail peptide comprising the amino acid        sequence CQRYVYS (SEQ ID NO: 1);    -   (b) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSIL (SEQ ID NO: 3);    -   (c) An isolated PAR1 c-tail peptide consisting of the amino acid        sequence SSECQRYVYSILCCK (SEQ ID NO: 4); and    -   (d) any fragment or modification of the peptides of (a) (b) or        (c).

In certain embodiments, the method further comprises administering anadditional therapeutic agent.

In another aspect, the present invention provides a method of inhibitingPAR2 mediated signal transduction comprising administering an agentcapable of selectively inhibiting the binding of PAR2 and a PH-domaincontaining protein, in particular Etk/Bmx.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount ofan agent capable of selectively inhibiting the binding of PAR2 and aPH-domain containing protein, in particular Etk/Bmx, or a pharmaceuticalcomposition comprising said agent to a patient in need thereof.

In certain embodiments said agent is a peptide.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount of apeptide, or a pharmaceutical composition comprising the peptide to apatient in need thereof, wherein the peptide is selected from the groupconsisting of:

-   -   (a) An isolated PAR2 c-tail peptide comprising the amino acid        sequence SHDFRDHA (SEQ ID NO: 2); and    -   (b) any fragment or modification of the peptide of (a).

In certain embodiments, the method further comprises administering anadditional therapeutic agent.

In accordance with certain embodiments, said disease is cancer.

In certain embodiments said cancer is selected from the group consistingof breast cancer, ovary cancer, lung cancer, colon cancer, prostatecancer and melanoma.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a peptide of the invention together with apharmaceutically acceptable carrier or diluent.

In a specific embodiment, the present invention provides apharmaceutical composition comprising a peptide of the inventiontogether with a pharmaceutically acceptable carrier or diluent for thetreatment of cancer.

The invention also encompasses a pharmaceutical composition comprising acombination of at least one isolated PAR1 c-tail peptide selected fromthe group consisting of:

-   -   (a) an isolated PAR1 c-tail peptide capable of interfering in        the binding reaction between PAR1 and a PH-domain containing        protein;    -   (b) an isolated PAR1 c-tail peptide comprising SEQ ID NO: 1;    -   (c) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSIL (SEQ ID NO: 3);    -   (d) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSILCCK (SEQ ID NO: 4); and    -   (e) any fragment or modification of the peptides of (a) (b) (c)        or (d).

and at least one isolated PAR2 c-tail peptide selected from the groupconsisting of:

-   -   (a) an isolated PAR2 c-tail peptide capable of interfering in        the binding reaction between PAR2 and a PH-domain containing        protein;    -   (b) an isolated PAR2 c-tail peptide comprising SEQ ID NO: 2; and    -   (c) any fragment or modification of the peptides of (a) or (b).

In a specific embodiment, the invention encompasses a pharmaceuticalcomposition comprising a combination of at least one isolated peptidecomprising SEQ ID NO: 1 or any fragment or modification thereof, and atleast one isolated peptide comprising SEQ ID NO: 2 or any fragment ormodification thereof.

In a specific embodiment the pharmaceutical compositions of theinvention are for the treatment of cancer. In yet other embodiments thepharmaceutical compositions further comprise an additional therapeuticagent.

The invention also concerns a peptide according to the above or apharmaceutical composition according to the above for use in combinationwith another therapeutic agent.

In another aspect the invention concerns an isolated peptide comprisingan amino acid sequence of the PAR1 cytoplasmic-tail for use as amedicament.

In yet another aspect the invention concerns an isolated peptidecomprising an amino acid sequence of the PAR2 cytoplasmic-tail for useas a medicament.

In another aspect, the present invention provides use of an agentcapable of selectively inhibiting the binding of PAR1 and a PH-domaincontaining protein, in the preparation of a pharmaceutical compositionfor treating a disease.

In one embodiment, said disease is cancer.

In another embodiment, said agent is a peptide.

In yet another aspect, the present invention provides use of a peptidein the preparation of a pharmaceutical composition for treating adisease, wherein the peptide is selected from the group consisting of:

-   -   (a) An isolated PAR1 c-tail peptide comprising the amino acid        sequence CQRYVYS (SEQ ID NO: 1);    -   (b) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSIL (SEQ ID NO: 3);    -   (c) An isolated PAR1 c-tail peptide consisting of the amino acid        sequence SSECQRYVYSILCCK (SEQ ID NO: 4); and    -   (d) any fragment or modification of the peptides of (a) (b) or        (c).

In one embodiment, the use further comprises administering an additionaltherapeutic agent.

In yet another aspect, the present invention provides use of an agentcapable of selectively inhibiting the binding of PAR2 and a PH-domaincontaining protein, in the preparation of a pharmaceutical compositionfor treating a disease.

In one embodiment said disease is cancer.

In another embodiment said agent is a peptide.

In another aspect, the present invention provides use of a peptide inthe preparation of a pharmaceutical composition for the treatment of adisease, wherein the peptide is selected from the group consisting of:

-   -   (a) An isolated PAR2 c-tail peptide comprising the amino acid        sequence SHDFRDHA (SEQ ID NO: 2); and    -   (b) any fragment or modification of the peptide of (a).

In one embodiment, the use further comprises administering an additionaltherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1A is a table listing the antibodies in an antibody array whichincludes thirty antibodies directed against various proteins. Theantibodies were immobilized on a membrane at a pre-determined positionas illustrated in the table. FIG. 1B is a photograph of the antibodyarray membrane showing specific protein-protein interactions betweenPAR1 and various signaling partners.

FIG. 2A is a schematic representation of the structure of various hPAR1constructs: wild-type hPar1 (wt), a truncated form (Truncated)representing the mutant hPar1 L369Z which lacks the entire cytoplasmictail, and Y397Z, another hPar1 deletion mutant which exhibits persistentsignaling due to impaired internalization. FIG. 2B demonstratessemi-quantitative RT-PCR analysis of cells transfected with varioushPar1 constructs (wt-hPar1, Truncated, Y397Z, and control (emptyvector)). Upper panel: PAR₁ N-terminus, middle panel: C-terminus. Allthe tested cells expressed similar GAPDH levels. FIG. 2C is a graphdemonstrating mouse mammary tumor growth in animals implanted with cellsexpressing wt hPar1 and variants. The graph demonstrates increase intumor volume (mm³, mean±SD) with time, for each of the tested groups: wthPar1 (▪), Y397Z hPar1 (□), truncated hPar1 (Δ), and empty vector (▴tumor volume 30±3 mm³*P<0.005). FIG. 2D is a photograph of tissuesections taken from orthotopic mammary fat pad tumors generated by MCF7cells over-expressing the various hPar1 constructs and stained withhematoxylin eosin (right panels). Magnification is ×100. Upper panel:Vector, second panel from the top: Truncated, third panel from the top:wild-type, lower panel: Y397Z.

FIG. 3A is a photograph showing sections of tumors generated by varioushPar1 constructs and subjected to immunohistochemical staining withKi-67 (upper panel), and with either an endothelial cell-specific lectin(α-lectin, mid-panel) or anti-CD31 (lower panel). FIG. 3B is a graphshowing the mean (±SD) number of Ki-67-positive cells per high powerfield (HPF) as counted in five microscope fields per tumor section. FIG.3C and FIG. 3D are graphs showing the mean (±SD) number of anti-lectin-or anti-CD31-stained cells, respectively, per high power field (HPF) ascounted in five microscope fields per tumor section. Error bars show+/−SD of mean and the P value was determined (** P<0.005 * P<0.001; Chi-square test). The data are representative of four independentexperiments performed in triplicates.

FIG. 4A shows Western blot analysis performed using anti-Shc antibodies,demonstrating binding of PAR₁ GST-C-tail to Shc adaptor. FIGS. 4B and 4Cshow co-immunoprecipitation analyses of PAR₁ and Shc. Lysates ofnon-activated or TFLLRNPNDK-activated MDA-MB-435 cells wereco-immunoprecipitated with either anti-PAR₁ (4B) or anti-Shc (4C)antibodies. FIG. 4D shows PAR₁ binding to the Shc-SH2 domain. MDA-MB-435cell lysates were loaded onto columns of GST-Shc-SH2, GST linked to atandem SH2 from a non-relevant protein, or GST alone. Specifically-boundproteins were eluted and detected with anti-PAR₁ antibodies. FIG. 4E isa schematic representation of the structure of PAR₁—C-tail. IL—internalligand; TM—transmembrane. Important tyrosine (Y) residues are indicatedand the conserved sequence is highlighted. FIG. 4F is a table showinganalysis of PAR₁ C-tail Y-residues by NetPhos 2.0 server. Y₃₈₁, Y₃₉₇ andY₄₂₀ were scored highly likely to undergo phosphorylation, as shown inthe table. “Pred” means “prediction” for the predicted score of each ofthe Y tyrosine residues that is relevant to phosphorylation.

FIG. 5A shows MRI analysis of tumors induced by injection of mouse CT-26colon carcinoma cells over-expressing wt hPar1, Y₃₈₁A hPar1 or emptyvector constructs. Tumor assessment was performed using T₂W fast SEimages (TR/TE=2000/40 ms). Representative axial liver sections of wthPar1 or Y₃₈₁A hPar1 CT-26-transfected cells, obtained at day 16, in theabsence (left panel) or presence (right panel) of SFLLRN, are seen.Liver margins are marked with a dashed line; lines mark tumor foci;scale bar represents a size of 1 cm and applies to all the images in A.FIG. 5B shows anatomical and histological examination of the tumors.Gross anatomical photos (Top) and H&E staining of liver sections(harvested on day 16) of activated wt hPar1 or Y₃₈₁A hPar1 CT-26 cells(mid and lower panels). Lines mark tumor foci; original magnification×100. FIG. 5C is a histogram of the number of liver metastases per mouseas measured by MRI. The experiments were performed in the presence orabsence of the PAR₁ agonist peptide (n=3-5 mice/group). X stands forsacrificed mice with overloaded liver tumors.

FIG. 6A is a gel demonstrating association between PAR₁ and Etk-PHdomain. Lysates of cells over-expressing Y397Z, wt or truncated hPar1,as well as a lysate of cells that do not express PAR₁ (e.g., JAR) and alysate of cells that express high levels of PAR₁ (e.g. the highlymetastatic MDA-435 cells) were applied to a GST-Etk-PH column.Specifically-bound proteins were detected using anti-PAR₁ antibodies.FIG. 6B is a gel demonstrating binding of Etk-PH domain with purifiedPAR₁ C-tail. PAR₁ C-tail was cleaved from the immobilized GST-C-tail,purified and re-applied onto a GST-Etk-PH domain column. FIG. 6C is agel demonstrating binding of GST-PAR₁ C-tail of wt and mutants. Lysatesof HEK293 cells transfected with either Etk/Bmx (A-D) or kinase-inactiveEtk/Bmx (KQ; E-G), were applied on various GST-PAR₁-C-tail columns(PAR₁-C-tail of wt, Y₃₈₁A, Y₃₈₃A) or GST-control column.Specifically-bound proteins were identified using anti-Etk/Bmxantibodies. Levels of GST were used as a control for protein loading.FIG. 6D is a photograph demonstrating immunohistological staining ofPAR₁ and Etk/Bmx on breast tissue biopsy specimens. Antibodies directedagainst PAR₁ (upper panel) or Etk/Bmx (lower panel) were applied tonormal and cancerous breast tissue specimens. The cancerous tissuesinclude DCIS (ductal carcinoma in situ), IDC (invasive ductal carcinoma)and lobular invasive carcinoma (lobular carcinoma). FIG. 7A is a geldemonstrating binding reactions between Etk/Bmx, Shc and PAR1 C-tail asdemonstrated by immunoprecipitation (IP). The IP was performed usinganti-PAR1 (ATAP, Santa Cruz, Calif.). FIGS. 7B and 7C are photographs ofgels showing peptide competition for PAR1 binding to GST-PH-Etk/Bmx. Inthe lower panel, a representative histogram shows the relativeintensities of the bands expressed as a ratio of PAR1 to GST-PH.

FIG. 8A is a histogram demonstrating the percentage of PAR1 surfaceexpression in cells transfected with empty vector, HA-wthPar1 andHA-hPar1 7A. FIG. 8B is a gel demonstrating staining with anti HAantibodies.

FIG. 9A is a schematic representation of wt hPar1 and the mutanthPar1-7A. FIG. 9B is a gel showing immunoprecipitation (IP) of PAR₁ withEtk/Bmx after activation in stable clones expressing either HA-tagged wthPar1 and/or a mutant construct of HA-hPar1-7A. The IP was carried outusing anti-HA antibodies. The Western blots were subjected to anti-Bmxfor the identification of Etk/Bmx-associated PAR₁. Levels of the HA-tag(for PAR₁) are shown in the middle panel. Similarly, levels of PAR₁ arealso shown by application of anti-PAR₁ (lower panel). The right sectionshows levels of plasmid transfection efficiencies in the cells, asindicated by HA-PAR₁ and Etk/Bmx analysis by Western blots. FIG. 9C is ahistogram showing numbers of invading cells (mean±SD) of ten fields perfilter in a Matrigel invasion assay. These data are representative ofthree experiments. FIG. 9D is a photograph showing MDA-MB-435 cellmonolayer scratched to introduce an equal gap-area. Control: controluntreated cells; TFLLRNPNDK-activated or SiRNA-Etk/Bmx andTFLLRNPNDK-activated. FIG. 9E is a gel showing RT-PCR analysis of thelevel of Etk/Bmx in MDA-MB-435 cells before and after SiRNA-Etk/Bmx cellinfection.

FIG. 10A-H are photographs showing morphogenesis of MCF10A spheroidsinfected with either wt hPar1, mutant hPar1-7A or with Etk/Bmx,maintained in 3-D Matrigel cultures. Fig. A-D show representativephase-contrast microscopic images of MCF10A cells under the followingconditions: A. control untreated MCF10A. B. MCF10A cells infected withEtk/Bmx and SFLLRNPNDK PAR₁-activated. C. MCF10A cells infected withboth wt hPar1 and Etk/Bmx and SFLLRNPNDK-activated. D. MCF10A cellsinfected with both mutant hPar1-7A and Etk/Bmx and SFLLRNPNDK-activated.Fig. E-H show representative confocal microscopic images of MCF10Aacini: E. Control untreated MCF10A, DAPI stained nuclei in arepresentative spheroid. F. MCF10A cells infected with both the mutanthPar1-7A and Etk/Bmx and SFLLRNPNDK-activated; DAPI staining of spheroidnuclei. G. MCF10A cells infected with both the wt hPar1 and Etk/Bmx andSFLLRNPNDK-activated; DAPI staining of the nuclei. H. MCF10A cellsinfected with both the mutant hPar1-7A and Etk/Bmx andSFLLRNPNDK-activated, stained for cell-cell contact withanti-E-cadherin.

FIG. 11A is a photograph showing tumors generated by cells transfectedwith various constructs. FIG. 11B is a histogram showing tumor weight.FIGS. 11C and 11D are photographs showing mice having tumors generatedby cells transfected with PAR1+Etk/Bmx (C) or PAR1A7+Etk/Bmx (D).

FIGS. 12A and 12B are photographs showing histological examination of atumor section (hematoxylin eosine staining). FIGS. 12C and 12D arephotographs of tissue sections of tumors produced by cells transfectedwith wtPAR1 & Etk/Bmx (12C) and tumors produced by cells transfectedwith PAR1 7A & Etk/Bmx (12D), stained with anti PCNA antibodies, anticaspase 3 antibodies and β-catenin antibodies.

FIG. 13A is a gel showing co-immunoprecipitation analyses using antiPAR2 antibodies and anti Etk/Bmx antibodies. FIG. 13B is a gel showingGST-PAR2C-tails: wt and deleted tails.

FIG. 14A is a gel showing the binding between Etk/Bmx expressing celllysates and GST-PAR2C-tails. FIG. 14B is a gel showing the bindingbetween Etk/Bmx expressing cell lysates and various truncatedGST-PAR2C-tails. Lanes: A. control, B. GST-PAR2C-tail wt; C.GST-PAR2C-tail K378Z; D. GST-PAR2C-tail K3356Z.

FIG. 15 is a schematic representation showing the sequence alignment ofthe PH domain of the proteins Etk/Bmx, vav3 and Akt.

FIG. 16A is a gel showing expression of Akt and Etk/Bmx in various celllines (CL1, HEK-293 and HCT-116). FIG. 16B is a gel showing bindingimmobilization of cell lysates following application on GST beads ofeither PAR₁-C-tail or PAR₂-C-tail. Lane A: GST column alone: Lanes B, C:HCT-116; Lanes D, E: HEK-293T+Etk.

FIG. 17A is a gel showing association of cell lysates of various celllines (containing HA-PAR1) with GST PH-Akt or GST-PH-Vav₃. FIG. 17B is agel showing the levels of expression of HA-PAR1 in the various celllines. FIG. 17C is a gel showing association of HA-PAR1 with GST PH-Aktor GST-PH-Vav₃ in HEK-293T cells transfected with increasingconcentrations of Etk/Bmx. FIG. 17D is a gel showing expression levelsof Etk/Bmx in Hek-293T cells transfected with Etk/Bmx.

FIG. 18A is a gel showing levels of co-immunoprecipitation between Aktand PAR1 at various time points following activation. FIG. 18B is a gelshowing levels of co-immunoprecipitation between Akt and PAR₁ at varioustime points following activation in cells transfected with either wthPar1 or with the mutant hPar1-7 A, incapable of binding Etk/Bmx. FIG.18C is a gel showing association between cell lysates expressing eitherwt hPar1 or the mutant hPar1 7A and GST-PH-Vav₃. FIG. 18D is a gelshowing expression of HA-tag in cells transfected with ha-PAR1 and incells transfected with the mutant PAR1-7A. FIG. 18E is a gel showingexpression of Akt in various cancer cells.

FIG. 19A is a gel showing association between PAR2 and PH-Akt or PH-Vav₃as demonstrated upon application of various cell lysates on GSTimmobilized PH-domain columns in the presence of absence of increasedconcentrations of Etk/Bmx. FIG. 19B is a gel showing expression levelsof Etk/Bmx.

FIGS. 20A and 20B are gels showing levels of binding between mutantversions of PAR2 c-tail (e.g., K352A, K349A, K356Z) and PH-Etk/Bmx (20A)or PH-Vav₃ or PH-Akt (20B). FIG. 20C shows PAR₂ expression levels. FIG.20D shows immunoprecipitation analyses before and after PAR₂ SLIGKVactivation of PAR₂ and Akt at 2 and 10 minutes following activation.FIG. 20E is a gel showing the association between PAR2 and Akt in wildtype PAR2 compared with the mutant form K352A.

FIG. 21A is a photograph showing MCF7 cells. FIGS. 21B and 21C showGFP-stained MCF7 cells.

FIG. 22 is a gel demonstrating the association between Etk/Bmx andPAR1-C-tail in the presence and absence of the GFP-labeled peptide.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention concerns isolated PAR1 cytoplasmic tail (c-tail)peptides and isolated PAR2 cytoplasmic tail (c-tail) peptides, as wellas compositions comprising these peptides, uses thereof and methods oftreating various diseases, in particular cancer.

The inventors of the present invention identified regions in thecytoplasmic portion of PAR1 and PAR2 that are responsible for signaltransduction, specifically, regions which are responsible for theinteraction with PH-domain containing proteins. The inventors alsodemonstrated that abrogation of the association between the cytoplasmicportion of PAR1 and PH-domain containing proteins (e.g. Etk) resulted ina reduction in PAR1 induced oncogenic activity, and that peptides of theinvention effectively penetrate cells and interfere in the associationbetween PAR1 and Etk.

Accordingly, the present invention includes peptides, pharmaceuticalcompositions and methods for alleviating pathological conditions whichare mediated by or associated with PAR1 or PAR2 cellular pathways. Inone embodiment the invention concerns peptides, pharmaceuticalcompositions and methods for treating cancer. Accordingly, a human oranimal with cancer is administered with a pharmaceutical compositioncomprising an isolated peptide comprising an amino acid sequence of thePAR1 or PAR2 cytoplasmic tail, or a fragment or modification thereof,wherein the isolated peptide interferes with the binding reactionbetween PAR1 or PAR2 and a PH domain containing protein. The peptidesmay be administered alone or in combination with other therapeuticagents for the amelioration and/or treatment of cancer. The othertherapeutic agents can be anti-angiogenic agents, anti-proliferativeagents, growth inhibitory agents (e.g. chemotherapeutic agents).

As used herein, “isolated” or “purified” when used in reference to apeptide means that the peptide has been removed from its normalphysiological environment (e.g. the peptide is present as such and notin the context of the complete protein, and not in its naturalcompartment, namely the peptide is isolated from the cell), or issynthesized in a non-natural environment (e.g. artificially synthesizedin a heterologous system).

As used herein the term “PAR1” refers to protease-activated receptor 1.As used herein the term “PAR2” refers to protease-activated receptor 2.PAR1 and PAR2 have an amino-terminal exodomain and a carboxy-terminalcytoplasmic or endodomain.

The terms “PAR1 cytoplasmic tail” or “PAR2 cytoplasmic tail” (alsoreferred to as “c-tail”, “cytoplasmic portion” or “cytoplasmic domain”)refer to the C-terminus (carboxy-terminus) of the PAR1 or PAR2 proteinwhich is present within the cell cytoplasm and contains the signalbinding region for downstream cellular signaling, as represented forexample by SEQ ID NO:33.

As used herein the terms “PAR1 cytoplasmic tail (c-tail) peptide” or“PAR2 cytoplasmic tail (c-tail) peptide” refer to peptides having aminoacid sequence that corresponds to portions of the cytoplasmic tail ofPAR1 or PAR2, respectively.

As used herein the term “fragment or modification” with respect to thepeptides of the invention refers to any variants or derivatives of thesepeptides.

The term “PH-domain containing protein” refers to proteins which includethe pleckstrin homology (PH) domain. Proteins which are involved insignal transduction consist of several modular domains. These modulescan confer catalytic or structural functions or mediate protein-proteininteractions. One of these module domains is the pleckstrin homology(PH) domain which is identified as a 100 to 120 amino acid stretch inmore than 250 human proteins (Rebecchi, M. J. and Scarlata, S. Annu RevBiophys Biomol Struct, 1998. 27: p. 503-28). Although the amino acidsequence of PH domains is not universally conserved, the tertiarystructure is remarkably conserved. PH-domain containing proteins can beidentified using various available proteomic databases, e.g. the ExPASyproteomics server. Non-limiting examples of PH-domain containingproteins are Etk/Bmx, Akt/PKB, Vav, SOS 1 and GAB 1.

The epithelial tyrosine kinase (Etk), also known as Bmx, is anon-receptor tyrosine kinase that is unique by virtue of being able tointeract with both tyrosine kinase receptors and GPCRs. This type ofinteraction is mainly attributed to the pleckstrin homology (PH) whichis followed by the Src homology SH3 and SH2 domains and a tyrosinekinase site.

Akt, also known as PKB, is a serine/threonine protein kinase that playsa pivotal role in multiple cellular processes and ubiquitously expressedin a wide spectrum of cell types.

The Vav₃ oncogene, a guanine nucleotide exchange factor (GEF) for theRho family GTPases, belongs to the Vav family proteins. The threemammalian proteins (Vav₁, Vav₂ and Vav₃) exhibit different tissuedistribution. While Vav₁ is primarily expressed in hematopoeitic cells,Vav₂ and Vav₃ are more ubiquitously expressed. Vav₃ has been shown to beover-expressed in human prostate cancer as well as breast cancer.

The term “interfere in the binding reaction between PAR1 and a PH-domaincontaining protein” (e.g. with Etk/Bmx) refers to a reduction orelimination of the association between PAR1 and a downstream signalingPH-domain containing protein. Such an association is usually inducedupon activation of PAR1. Similarly, the term “interfere in the bindingreaction between PAR2 and a PH-domain containing protein” (e.g. withEtk/Bmx) refers to a reduction or elimination of the association betweenPAR2 and a downstream signaling PH-domain containing protein. Such anassociation is usually induced upon activation of PAR2. The ability ofan agent to interfere in these binding reactions can be determined bymethods known in the art. Some of these methods are exemplified in theExamples provided below.

The terms “pathological condition” or “disease” are commonly recognizedin the art and designate the presence of at least one sign and/orsymptom in a subject or a patient that are generally recognized asabnormal. Pathological conditions or diseases may be diagnosed andcategorized based on pathological changes. Signs may include anyobjective evidence of a disease such as changes that are evident byphysical examination of a patient or the results of diagnostic teststhat may include, among others, laboratory tests. Symptoms aresubjective evidence of disease or a patient condition, e.g. thepatient's perception of an abnormal condition that differs from normalfunction, sensation, or appearance, which may include, withoutlimitations, physical disabilities, morbidity, pain and other changesfrom the normal condition experienced by a subject.

Pathological conditions or diseases which are mediated by or associatedwith PAR1 or PAR2 cellular pathways include but are not limited to,cancer, acute and chronic inflammatory diseases, for exampleinflammatory diseases of the joints (e.g. arthritis), lungs (e.g.respiratory tract disorders such as pulmonary fibrosis, asthma andchronic obstructive pulmonary disease), brain, gastrointestinal tract(e.g. inflammatory bowel disease, such as colitis) and vascular systems(including cardiovascular diseases, e.g. thrombosis and restenosis), aswell as inflammation associated with tissue response to injury. Alsoincluded are wound healing and pain, as well as allergies such asallergic contact dermatitis, atopic dermatitis and pruritus.

As used herein the term “Cancer” refers to any type of malignantproliferative disease and is used as commonly known in the art. Inparticular, the present invention concerns cancer types which areassociated with PAR1 and/or PAR2 signal transduction and which canbenefit from interfering with PAR1 and/or PAR2 signal transduction. Nonlimiting examples include breast cancer, ovary cancer, lung cancer,colon cancer, prostate cancer and melanoma.

The term “treating” refers to a reduction or elimination of at least onesign and/or symptom of a specific disease or condition. The term alsoencompasses prevention or attenuation of disease progression.

The term “agent” or “therapeutic agent” as used herein refers to achemical entity or a biological product, or combination of chemicalentities or biological products, which are used to treat, prevent orcontrol a disease or a pathological condition.

As used herein, the term “therapeutically effective” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment.

Thus, in connection with the administration of an agent or apharmaceutical composition, an agent or a pharmaceutical compositionwhich are “effective against” a disease or pathological conditionindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as an improvement of symptoms, a cure, a reduction indisease signs or symptoms, extension of life, improvement in quality oflife, or other effect generally recognized as positive by medicaldoctors familiar with treating the particular type of disease orcondition.

The term “therapeutically effective amount” refers to a dosage or amountthat is sufficient to reduce, halt, or slow disease progression toresult in alleviation, lessening or amelioration of symptoms in apatient or to achieve a desired biological outcome, e.g. slow or stoptumor growth or reduction or disappearance of a tumor.

As used herein the term “patient” relates to a subject suffering from orsuspected of suffering from a specific disease or pathological conditionor presents with at least one sign of a specific disease or pathologicalcondition. Preferably, the subject is a human subject.

“Pharmaceutically acceptable carriers or diluents” also referred to aspharmaceutically acceptable excipients or vehicles refer to apharmaceutically acceptable material, composition or vehicle, suitablefor administering compounds of the present invention to mammals,specifically to humans. These include for example, water, saline,glycerol, ethanol etc. Additionally, auxiliary substances, such aswetting or emulsifying agents, buffers, preservatives, anti-oxidants,surfactants, and the like, may be present in such carriers or diluents.

Examples of suitable “buffers” include Tris, Hepes, triethanolamine,histidine, or any others known in the art.

“Preservatives” can act to prevent bacteria, viruses, and funghi fromproliferating in the pharmaceutical composition or formulation, andanti-oxidants can function to preserve the stability. Examples includeoctadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride,benzalkonium chloride, and benzethonium chloride. Other types ofcompounds include aromatic alcohols such as phenol and benzyl alcohol,alkyl parabens such as methyl or propyl paraben, and m-cresol.

A “surfactant” can act to decrease turbidity or denaturation of aprotein or a peptide in a pharmaceutical composition or formulation.Examples of surfactants include non-ionic surfactant such as apolysorbate, e.g. polysorbates 20, 60, or 80, a poloxamer, e.g.poloxamer 184 or 188, Pluronic polyols, ethylene/propylene blockpolymers or any others known in the art.

The peptides of the invention or the pharmaceutical compositions of theinvention may be used in combination with other therapeutic agents. “Usein combination” as used herein refers to the administration with atleast one other therapeutic agent, either at the same time, in the samecomposition, at alternating times (prior to or subsequent to), inseparate compositions, or combinations thereof.

The inventors have identified PAR1 and PAR2C-tail as a scaffold site forthe interaction with signaling partners. In addition to identifying keypartners, the hierarchy of binding was determined and a specific regionin PAR1C-tail was identified as critical for breast cancer signaling.Specifically, Etk/Bmx and Shc were found to form a physical complex withPAR1C-tail. Etk/Bmx was shown to bind to PAR1-C-tail via its PH domainenabling the subsequent association of Shc. The physiologicalsignificance of PAR1-Etk/Bmx binding is emphasized by inhibition ofMatrigel invasion and appearance of nearly intact acini morphogenesis ofcell architecture when this site is mutated to abrogate the binding ofEtk/Bmx. The use of consecutive A residues inserted into the proposedEtk/Bmx binding region of PAR1C-tail (e.g., hPar1-7A) abolishedPAR1-induced pro-oncogenic properties. Thus, by preventing the bindingof a key signaling partner to PAR1C-tail, efficient inhibition ofPAR1-induced tumor-associated functions, including loss of epithelialcell polarity, migration and invasion through basement membranes, isobtained.

The inventors also identified an amino acid sequence at the C-tail ofPAR2 which is responsible for the interaction with Etk/Bmx as well asadditional PH-domain containing proteins.

In one of its aspects, the present invention thus provides isolatedpeptides corresponding to the PAR1 cytoplasmic tail or any fragmentthereof, more specifically peptides corresponding to the“signal-binding” region in the cytoplasmic tail. The PAR1-Etk/Bmxbinding motif sequence was found to be: NH2-CQRYVYS-COOH. Therefore, inone embodiment the present invention provides a peptide comprising theamino acid sequence CQRYVYS (SEQ ID NO: 1) or any fragment ormodification thereof that maintains the ability to interfere in thebinding reaction between PAR1 and Etk/Bmx.

In one embodiment, the present invention provides a peptide having 7 ormore, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 ormore, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 ormore, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, and 25or more amino acids comprising the amino acid sequence CQRYVYS or anyfragment or modification thereof.

In one embodiment, the present invention provides a peptide havingbetween about 7 and about 25 amino acids comprising the amino acidsequence CQRYVYS or any fragment or modification thereof.

In one embodiment said peptide is 12 amino acids long. In a specificembodiment said peptide consists of the sequence NH2-SSECQRYVYSIL-COOH(SEQ ID NO: 3), or any fragment or modification thereof that maintainsthe ability to interfere in the binding reaction between PAR1 andEtk/Bmx. In another embodiment said peptide is 15 amino acids long. In aspecific embodiment said peptide consists of the sequenceNH2-SSECQRYVYSILCCK-COOH (SEQ ID NO: 4) or any fragment or modificationthereof that maintains the ability to interfere in the binding reactionbetween PAR1 and Etk/Bmx.

Without wishing to be bound by theory, longer peptides (i.e. peptideslonger than 7 amino acids, or longer than 8 amino acids, or longer than9 amino acids, or longer than 10 amino acids, or longer than 11 aminoacids) may have preferable characteristics such as better solubility orstability as compared with shorter peptides (e.g. peptides having 7amino acids or 8 amino acids).

In another of its aspects, the present invention provides isolatedpeptides corresponding to the PAR2 cytoplasmic-tail, or any fragmentthereof, more specifically peptides corresponding to the“signal-binding” region in the cytoplasmic tail. The PAR2-Etk/Bmxbinding motif sequence was found to be: NH2-SHDFRDHA-COOH. In oneembodiment the present invention provides a peptide having 8 or more, 9or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21or more, 22 or more, 23 or more, 24 or more, and 25 or more amino acidscomprising the amino acid sequence SHDFRDHA or any fragment ormodification thereof that maintains the ability to interfere in thebinding reaction between PAR2 and Etk/Bmx.

In one embodiment, the present invention provides a peptide havingbetween about 8 and about 25 amino acids comprising the amino acidsequence SHDFRDHA (SEQ ID NO: 2) or any fragment or modificationthereof.

In one embodiment said peptide is 15 amino acids long.

Preferably, the peptides of the invention are designed so as topenetrate cell membranes. In certain embodiments, cell penetratingmoieties or membrane tethering moieties may be attached to the peptidesof the invention in order to allow cell membrane penetration. Cellpenetrating moiety is a compound which mediates transfer of a substancefrom an extracellular space to an intracellular compartment of a cell.Cell penetrating moieties shuttle a linked substance into the cytoplasmor the cytoplasmic space of the cell membrane. Membrane tetheringmoieties are compounds which associate or bind to a cell membrane. Thusthe membrane tethering moiety brings the substance to which themembrane-tethering moiety is attached in close proximity to the membraneof a target cell. For example, cell penetrating moieties or membranetethering moieties may be hydrophobic moieties. A cell penetratingmoiety and a membrane tethering moiety includes, but is not limited to,a lipid, cholesterol, phospholipids, steroid, or a fatty acid moiety.Cell penetrating moieties may also be Cell Penetrating Peptides (CPP)such as Tat (trans-activating transcriptional activator from HIV-1) orPenetratin™ (Penetratin™ 1 is a 16-amino acid peptide corresponding tothe third helix of the homeodomain of Antennapedia protein), or smallmolecule synthetic analogues of CPP. The cell penetrating moiety ormembrane tethering moiety is attached to the C-terminal amino acid, theN-terminal amino acid, or to an amino acid between the N-terminal andC-terminal amino acid of the peptide of the invention.

The peptides of the invention may also be modified in manners whichincrease their solubility, stability or half-life in the body (e.g.modifications which reduce proteolytic degradation of the peptide uponadministration to the patient, or reduce the clearance from the blood).These modifications include, but are not limited to association withstabilizing molecules, e.g. pegylation, encapsulation (for example inlyposomes) using methods well known in the art.

The present invention also encompasses peptidomimetics, i.e. anypeptides or other types of agents which mimic the activity of thepeptides of the invention. The present invention is thereforecontemplated to include any variants, derivatives, fragments ormodifications of the peptides of the invention. These variants,derivatives, fragments or modifications maintain the activity of theoriginal peptide. In certain embodiments, the variants, derivatives,fragments or modifications of the peptides of the invention are activein vivo or in vitro in interfering in the binding reaction between PAR1or PAR2 and a PH-domain containing protein, e.g. Etk/Bmx.

In certain embodiments the variants, derivatives, fragments ormodifications are biologically active and have at least about 80% aminoacid sequence identity, more preferably at least about 90% sequenceidentity, and even more preferably, at least 95%, 96%, 97%, 98%, or 99%sequence identity with any one of the above recited PAR1 or PAR2 c-tailpeptides.

“Percent (%) amino acid sequence identity” with respect to the sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe PAR1 or PAR2 c-tail peptides sequences, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Alignment for the purposes of determiningpercent amino acid sequence identity can be achieved in various waysthat are well known in the art.

Accordingly, various modifications may be made in the peptides of theinvention. Mutations may be inserted in the identified binding region bysite-directed mutagenesis e.g. by using QuickChange kit (Stratagene, LaJolla, Calif.). The binding capabilities of naïve wt protein can then becompared with proteins comprising the mutated domain.

For example, an alanine scan can be used to identify the residues thatare critical for PAR1 “signal binding”. Different peptides are producedeach having an alanine residue in a different position, for example asfollows:

(SEQ ID NO: 5) NH2-SSECQRYVYSILCC A -COOH (SEQ ID NO: 6)NH2-SSECQRYVYSILC A K-COOH (SEQ ID NO: 7) NH2-SSECQRYVYSIL A CK-COOH(SEQ ID NO: 8) NH2-SSECQRYVYSI A CCK-COOH (SEQ ID NO: 9) NH2-SSECQRYVYSA LCCK-COOH (SEQ ID NO: 10) NH2-SSECQRYVY A ILCCK-COOH (SEQ ID NO: 11)NH2-SSECQRYV A SILCCK-COOH (SEQ ID NO: 12) NH2-SSECQRY A YSILCCK-COOH(SEQ ID NO: 13) NH2-SSECQR A VYSILCCK-COOH (SEQ ID NO: 14) NH2-SSECQ AYVYSILCCK-COOH (SEQ ID NO: 15) NH2-SSEC A RYVYSILCCK-COOH(SEQ ID NO: 16) NH2-SSE A QRYVYSILCCK-COOH (SEQ ID NO: 17) NH2-SS ACQRYVYSILCCK-COOH (SEQ ID NO: 18) NH2-S A ECQRYVYSILCCK-COOH(SEQ ID NO: 19) NH2- A SECQRYVYSILCCK-COOH

Next, in order to improve the peptide inhibitory activity thenon-essential residues identified in the ala scan can be replaced withother amino acid residues, optionally by non-natural amino acids.

The following are non-limiting examples of mutations that may beinserted in PAR2: wt C-tail-SHDFRDHAZ (SEQ ID NO:20); mutated forms ofPAR2C-tail-SAAARDHAZ (SEQ ID NO:21); or SHDFAAAAZ (SEQ ID NO:22). Forthese exemplary mutations the following primers may be used:

Primer1: (SEQ ID NO: 23) F-cccctttgtctattactttgtttcagctgctgccagggatcatg;(SEQ ID NO: 24) R-gcatgatccctggc agcagctgaa acaaagtaa tagacaaagggg;Primer2: (SEQ ID NO: 25)F-cacatgatttcgcggctgc tgcaaagaacgctc tcctttgccg; (SEQ ID NO: 26)R-cggcaaag gagagcgttctttgcagca gccgcgaaatcatgtg;

Chemical modifications can also be made in the peptides. For example,relevant amino acids (e.g., leucine, isoleucine) may be modified toinclude alternative side chains, non-limiting examples of such sidechains include: ethyl, n-butyl, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CHOHCH₃ and—CH₂SCH₃. Tyrosine amino acid can be modified by having substitutedbenzyl or phenyl side chains. Preferred substituents include one or moreof the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and—CN.

Glutamic acid may be modified to substituted or unsubstituted aliphatic,aromatic or benzylic ester of glutamic acid (e.g., methyl, ethyl,n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl),glutamine, CO—NH-alkylated glutamine or asparagine (e.g., methyl, ethyl,n-propyl and iso-propyl) and modified amino acids having the side chain—(CH₂)₃COOH, an ester thereof (substituted or unsubstituted aliphatic,aromatic or benzylic ester), an amide thereof and a substituted orunsubstituted N-alkylated amide thereof.

For lysine or arginine amino acids modification will be carried out by 1to about 3 additional methylene units in the side chain.

The amino acids serine and cysteine may be modified having C₁-C₅straight or branched alkyl side chains substituted with —OH or —SH.

The term “chemical modification” as used herein includes modification atthe side chain of the amino acid residue, as well as modification of thepeptidic bond. Accordingly, a functional group may be added to the sidechain, deleted from the side chain or exchanged with another functionalgroup. Typically, the modifications are conservative modificationsresulting in conservative substitution. Examples of conservativemodifications of this type include adding an amine or hydroxyl,carboxylic acid to the aliphatic side chain of valine, leucine orisoleucine, exchanging the carboxylic acid in the side chain of asparticacid or glutamic acid with an amine or deleting the amine group in theside chain of lysine or ornithine. Other chemical modifications known inthe art include arboxymethylation, acylation, phosphorylation,glycosylation or fatty acylation, and others.

The “chemical modification” also includes alteration of a bond withinthe peptidic backbone, i.e. that the bond between the N— of one aminoacid residue to the C— of the next has been altered to non-naturallyoccurring bonds by reduction (to —CH₂—NH), alkylation (methylation) onthe nitrogen atom, or the bonds have been replaced by amidic bond, ureabonds, or sulfonamide bond, etheric bond (—CH₂—O—), thioetheric bond(—CH₂—S—), or to —C—S—NH—; The side chain of the residue may be shiftedto the backbone nitrogen to obtain N-alkylated-Gly (a peptidoid).Modification also includes cyclization of the amino acid molecule, e.g.by forming S—S bonds. S—S bonds may be formed via the inclusion ofsulphor-containing amino acid residues, such as cysteine at eachterminus of the amino acid molecule. Cyclic peptides have been shown tobe more stable and with higher biological activity than thecorresponding linear molecule (Jining L. et al. Eur. J. Biochem271:2873-2886 (2004)).

The peptides of the invention may be provided in a soluble or alyophilized (dry) form.

The peptides of the invention can be prepared by automated peptidesynthesis methodologies well known in the art.

Alternatively the PAR1 or PAR2 c-tail peptides of the invention may beisolated from the complete PAR1 or PAR2 protein, respectively,preferably, the human PAR1 or PAR2 protein. Isolation from the completePAR1 or PAR2 protein can be effected, for example, by proteolysis ofPAR1 or PAR2 by proteases such as thrombin, plasmin, activated proteinC, or metalloprotease-1.

Alternatively, the PAR1 or PAR2 c-tail peptides of the invention may beproduced recombinantly using methods well known in the art.

In another aspect, the present invention provides a method of inhibitingPAR1 mediated signal transduction comprising administering an agentcapable of selectively inhibiting the binding of PAR1 and a PH-domaincontaining protein, e.g. Etk/Bmx, vav3 or Akt.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount ofan agent capable of selectively inhibiting the binding of PAR1 and aPH-domain containing protein, or a pharmaceutical composition comprisingsaid agent.

In a specific embodiment the PH-domain containing protein is Etk/Bmx. Inother embodiments the PH-domain containing protein is vav3 or Akt.

The invention is not limiting with respect to the type of agent thatinhibits or interferes with the binding between PAR1 and the PH-domaincontaining protein. The agent may be, but is not limited to, a peptide,a peptidomimetic molecule, a polypeptide, or a small molecule (e.g. achemical compound). In a specific embodiment the agent is a peptide.

The invention further provides a method of treating a disease comprisingadministering a therapeutically effective amount of a peptide, or apharmaceutical composition comprising the peptide, to a patient in needthereof, wherein the peptide is selected from the group consisting of:

-   -   (a) An isolated PAR1 c-tail peptide comprising the amino acid        sequence CQRYVYS (SEQ ID NO: 1);    -   (b) an isolated PAR1 c-tail peptide consisting of the sequence        SSECQRYVYSIL (SEQ ID NO: 3);    -   (c) An isolated PAR1 c-tail peptide consisting of the amino acid        sequence SSECQRYVYSILCCK (SEQ ID NO: 4); and    -   any fragment or modification of the peptides of (a) (b) or (c).

PAR1 has been shown to play a central role in the invasive andmetastatic cancers of breast, ovaries, lung, colon, prostate andmelanoma. Therefore, in a specific embodiment the invention is directedto a method of treating cancer.

In certain embodiments the cancer is selected from the group consistingof breast cancer, ovary cancer, lung cancer, colon cancer, prostatecancer and melanoma.

In another aspect, the present invention provides a method of inhibitingPAR2 mediated signal transduction comprising administering an agentcapable of selectively inhibiting the binding of PAR2 and a PH-domaincontaining protein, e.g. Etk/Bmx, vav3 or Akt.

In another aspect, the present invention provides a method of treating adisease comprising administering a therapeutically effective amount ofan agent capable of selectively inhibiting the binding of PAR2 and aPH-domain containing protein, or a pharmaceutical composition comprisingsaid agent.

In a specific embodiment the PH-domain containing protein is Etk/Bmx. Inother embodiments the PH-domain containing protein is vav3 or Akt.

The invention is not limiting with respect to the type of agent thatinhibits or interferes with the binding between PAR2 and the PH-domaincontaining protein. The agent may be, but is not limited to, a peptide,a peptidomimetic molecule, a polypeptide, or a small molecule (e.g. achemical compound). In a specific embodiment the agent is a peptide.

The invention further provides a method of treating a disease comprisingadministering a therapeutically effective amount of a peptide, or apharmaceutical composition comprising the peptide to a patient in needthereof, wherein the peptide is selected from the group consisting of:

-   -   (a) An isolated PAR2 c-tail peptide comprising the amino acid        sequence SHDFRDHA (SEQ ID NO: 2); and    -   (b) any fragment or modification of the peptide of (a).

In a specific embodiment the invention is directed to a method oftreating cancer.

In certain embodiments the cancer is selected from the group consistingof breast cancer, ovary cancer, lung cancer, colon cancer, prostatecancer and melanoma.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a peptide of the invention together with apharmaceutically acceptable carrier or diluents.

In another embodiment, the present invention provides use of a peptideof the invention in the preparation of a pharmaceutical composition.

In certain embodiments said pharmaceutical composition is for thetreatment of cancer.

In one embodiment the present invention provides a combination of PAR1c-tail peptides and PAR2 c-tail peptides of the invention. Thiscombination of peptides may be used as a therapeutic agent for thetreatment of cancer.

Any of the peptides, pharmaceutical compositions or methods of theinvention may used in combination with additional therapeutic agents,e.g. for the treatment of cancer.

Example 1 Materials and Methods

Cell culture: MCF7 and MDA-MB-435 human breast carcinoma, CT-26 mousecolon carcinoma, HEK-293 cells and the African green monkey kidneyfibroblast cell line COS 1 (obtained from the ATCC, VA, USA) weremaintained in DMEM with 10% fetal calf serum. Stable clonal cell linesover-expressing wt hPar1, Y397Z hPar1, truncated hPar1 and Y/A mutants;Y₃₈₁A&₃₈₃A hPar1 or the wt-Etk and Etk-KQ were selected for G418resistance (800 μg/ml).

Plasmids and transfection: MCF7 cells were transfected with 1-2 μg ofeither wt human hPar1 or truncated hPar1 or Y397Z hPar1 cDNA, or with acontrol pcDNA3 vector (Invitrogen, Carlsbad, Calif.) using FuGenetransfection reagent (Roche Molecular Biochemicals, Indianapolis, Ind.).Transfected cells were selected with G418 (800 μg/ml) to obtain stablepopulations of cells expressing hPar1 and the variants. Etk/Bmx plasmids(e.g., wt, kinase-dead, KQ and GST-PH-Etk/Bmx) (Tsai Y T, et al. (2000)Mol Cell Biol. 20:2043-2054.) were transfected into HEK-293 cells usingthe same protocol as previously described.

RNA isolation and RT-PCR. RNA was isolated with Tri-Reagent (MRC,Cincinnati, Ohio) according to the manufacturer's instructions. Afterreverse transcription of 1 μg total RNA by oligo (dT) priming, cDNA wasamplified using Taq DNA polymerase (Promega, Madison, Wis.). Comparativesemi-quantitative PCR was performed using the following primers: GAPDHsense: 5′-CCA CCC ATG GCA AAT TCC ATG GC-3′ (SEQ ID NO: 27) andantisense: 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′ (SEQ ID NO: 28)primers. PAR₁ N-terminus primers were as follows: hPar1-sense:5′-CTCGTCCTCAAGGAGCAAAC-3′ (SEQ ID NO: 29), antisense orientation:5′-TGGGATCGGAACTTTCTTTG-3′ (SEQ ID NO: 30) (resulting in a 564-bp PCRproduct). PAR₁ C-tail primers-sense: 5′-TAC TAT TAC GCT GGA TCC TCTGAG-3′ (SEQ ID NO: 31) and antisense: 5′-CTT GAA TTC CTA AGT TAACAGCTT-3′ (SEQ ID NO: 32). These primers give rise to a 181-bp productcorresponding to the entire PAR₁ C-tail site, as follows:

(SEQ ID NO: 33) YYYASSECQRYVYSILCCKESSDPSYNSSGQLMASKMDTCSSNLNNSI YKKLLT.

Animal studies: Mammary gland model. Female athymic nude mice at 6-8weeks of age were pre-implanted subcutaneously with pellets containing1.7 mg β-estradiol (60-day release, Innovative Research of America,Sarasota, Fla.). Mouse mammary fat pads were then injected with 1×10⁷MCF-7 cells stably transfected with hPar1 wt and mutant constructs(e.g., Y397Z and truncated) or pcDNA3 control plasmid. Mice weremonitored for tumor size by external caliber measurements (length andwidth) on days 10, 22, 25, 29, 33, 36 and 45. Tumor volume (V) wascalculated by V=L×W2×0.5, where L is length and W is width. On day 45,mice were sacrificed and tumors were removed, weighed and fixed informalin for histology. All animal experiments were approved by theanimal committee of the Hebrew University, Jerusalem, Israel(MD-107.05-4).

Liver metastasis model: CT-26 mouse colon carcinoma cells were stablytransfected with either wt hPar1 or hPar1-Y₃₈₁A constructs. Theactivation of PAR₁ (using the peptide SFLLRN) was performed prior toinjection into the mice. CB6F1 mice were anesthetized (75 mg/kgketamine+3 mg/kg xylazine, i.p.), and the spleen was exteriorizedthrough an incision (1.0 cm) on the left side of the mouse. CT-26 cells(10⁴ cells/mouse) transfected with the different constructs (e.g.,hPar1, hPar1 Y381A, mock-transfected vector) were injected into thespleen using a 30-gauge needle. The cell suspension was allowed to enterthe portal circulation over a short period (5 minutes), after which thespleen was removed, as previously described (Kuruppu D, et al (2002) JSurg Res. 103:47-54.). The wound was sutured and the animal was allowedto recover. MRI images were monitored every 2-3 days on a 4.7T BrukerBiospec spectrometer using a bird-cage coil. Tumor assessment was madeby serial coronal and axial T₂W fast SE images (TR/TE=2000/40 ms). Allexperiments were performed in accordance with the guidelines of theAnimal Care and Use Committee of the Hebrew University, Jerusalem,Israel (MD-107.05-4).

PAR₁ activation: PAR₁ was activated by theSFLLRN(H-Ser-Phe-Leu-Leu-Arg-Asn-NH₂) peptide (SEQ ID NO: 34), theTFLLRNPNDK peptide (SEQ ID NO: 35), a selective PAR₁ agonist, orthrombin (1 U/ml).

Histology: Tissue samples derived from the primary tumors were fixedwith 4% formaldehyde in PBS, embedded in paraffin and sectioned (5-μmsections). After de-paraffinization and re hydration, sections werestained with hematoxylin and eosin (H&E) or subjected toimmunohistochemistry using specific antibodies.

Histological evaluation and scoring: The combined histological resultswere assessed and scored as previously described (Groeger A M, et al.(2004) Histopathology 44:54-63). The measurements per slide section werecarried out using anatomical compartments, using an ocular micrometer(WHIOX2, Olympus, N.J., USA). Slides review was independently performedby two investigators (BM and RB). Discrepancies were resolved bysimultaneous re-examination of the slides by both investigators using adouble-headed microscope. The microscope was calibrated with amicrometer slide before each measurement. All measurements wereperformed on the monitor screen using a x40 objective. On examining thesections for selection of fields tumor cells from the most cellular areaat the center of the tumor were selected. Necrotic and inflammatory areawere avoided. Eight microscopic fields were screened, 10 cells/fieldwere selected and no less than 50 cells/tumor case were assessed. Thepositive rate of staining is expressed as a mean±SD per tumorhistological subtype from selected cases.

Immunohistochemistry. Sections were subjected to inactivation ofendogenous peroxidase (3% H₂O₂ in DDW), antigen retrieval by microwaveoven (3 min) in citrate buffer (0.01 M, pH 6.0), and blocking with 10%goat serum in PBS. Sections were then incubated with antibodies directedagainst Von-Willebrand factor (anti-factor VIII, DAKO, Carpinteria,Calif.), Ki-67 (Clone SP6, Lab Vision-NeoMarkers, Fremont, Calif.), oran endothelial cell-specific lectin (Bandeiraea simplicifolia BS-1isolation), followed by incubation with horseradishperoxidase-conjugated anti-rabbit antibody (DAKO, Carpinteria, Calif.).Color was developed by incubation (10 min) with the Zymed AEC substratekit (Zymed Laboratories, South San Francisco, Calif.), andcounterstained with Mayer's hematoxylin.

Preparation of hPar1 constructs: truncated hPar1, Y397Z hPar1, Y₃₈₁AhPar1 and Y383A hPar1. Detection of hPar1 was carried out using primers:sense orientation: 5′-CTCGTCCTCAAGGAGCAAAC-3′ (SEQ ID NO: 29), antisenseorientation: 5′-TGGGATCGGAACTTTCTTTG-3′ (SEQ ID NO: 30). For thePAR₁-C-tail primers: sense orientation: 5′-TACTATTACGCTGGATCCTCTGAG-3′(SEQ ID NO: 31), antisense: 5′-(SEQ ID NO: 33).

Using polymerase chain reaction, a PAR-1 mutant protein truncated in itscytoplasmic tail after amino acid leucine 369 or at tyrosine 397 wasconstructed. Y397Z construct: PAR-1 cDNA served as a template foramplifying the fragment containing STOP codon using the followedprimers: sense: 5′-ATA AGC ATT GAC CGG TTT CTG-3′ (SEQ ID NO: 36) andantisense: 5′-GCT CTA GAT TTT AAC TGC TGG GAT CGG AAC-3′ (SEQ ID NO:37). Replacement of tyrosine residues at PAR₁ cytoplasmic tail wasachieved using specific primers containing the point mutation. Primersequences were as follows: 381-sense: 5′-TGC CAG AGG GCT GTC TAC AGT ATCTTA TGC-3′ (SEQ ID NO: 38), 381-antisense: 5′-GAT ACT GTA GAC AGC CCTCTG GCA CTC AGA-3′ (SEQ ID NO: 39), 383-sense: 5′-GCC AGA GGT ACG TCGCAA GTA TCT TAT GCT GCA AA-3′ (SEQ ID NO: 40), 383-antisense: 5′-AAG ATACTT GCG ACG TAC CTC TGG CAC TCA G-3′ (SEQ ID NO: 41). The amplified DNAfragment was digested with XbaI and HindIII from PAR₁ cDNA and clonedinto a pcDNA3 plasmid, followed by DNA sequencing. To confirm thefunctional integrity of the DNA constructs, wt and mutant cDNAs weretransiently expressed in COS-1 cells that were subsequently subjected toFACS analysis with a PAR-1-specific antibody (WEDE15-PE, Immunotech,Cedex, France).

HA-tag wt hPar1 and HA-mutant hPar1-7A C-tail constructs. The mutantswere designed for insertion of A at the carboxy terminus of PAR₁residues 378-384: SSECQRYVYSILCC (SEQ ID NO: 42) to SSEAAAAAAAILCC(named hPar1-7A mutant) (SEQ ID NO: 43). For HA-tag wt hPar1 constructPCR primers were designed and added downstream to the ATG start codon.Primers for the HA-tag are as follows: sense: 5′-TAC CCA TAC GAT GTT CCAGAT TAC GCT-3′ (SEQ ID NO: 44) and anti-sense: 5′-AGC GTA ATC TGG AACATC TA TGG GTA-3′ (SEQ ID NO: 45). Replacement of seven residues withAla (A) at positions 378-384 was made by synthesis of oligos containingthe mutation. Primer sequences were as follows: hPar1 7A mutant: sense:5′-TCT GAG GCT GCT GCT GCT GCT GCA GCT ATC TTA-3′ (SEQ ID NO: 46) andanti-sense: 5′-TAA GAT AGC TGC AGC AGC AGC AGC AGC CTC AGA-3′ (SEQ IDNO: 47). PCR products were then used as primers on an hPar1 cDNAtemplate to create an extended product of introduced mutations into thefull-length sequence. The amplified DNA fragment was digested with PinAIand XbaI from PAR₁ cDNA and cloned into pcDNA3-hPar1 plasmid followed byDNA sequencing.

GST-C-tail cloning. GST-C-tail of PAR₁ fragment, containing 54 aminoacids from serine 369 to residue 425, was prepared using RT-PCR (5′-TACTAT TAC GCT GGA TCC TCT GAG-3′ (SEQ ID NO: 48) and 5′-CTG AAT TCC TAAGTT AAC AGC TT-3′ (SEQ ID NO: 49)). The resulting DNA fragment wasfurther cut with the appropriate restriction enzymes (BamHI and EcoR1)and ligated into pGEX2T vector. The GST-C-tail was separated bySDS-PAGE, which indicated that the fusion protein of the C-tail wasadequately prepared. The molecular weight of GST protein is 27 kD andthe GST-C tail fusion protein is 32 kD. GST-Shc-SH2 and tandem SH2 werekindly provided by S. Katzav, Hubert H. Humphrey Center for ExperimentalMedicine and Cancer Research, Hebrew University-Hadassah Medical School,Jerusalem.

GST fusion protein columns. Fusion proteins were purified fromtransformed Escherichia coli bacteria that had been stimulated withisopropyl-β-D-thio-galactopyranoside (IPTG) at a concentration of 0.3μM. Bacteria were lysed according to published procedures, and thenimmobilized on glutathione Sepharose beads (Pharmacia). Briefly,MDA-MB-435 cell lysates were applied to GST-PAR1C-tail or GST controlcolumns. After 2 h binding periods to the designated protein/s celllysates to the columns, a washing step was performed. The washes (×3)were carried out using a “wash buffer” including: 100 mM NaCL, 20 mMEDTA, 10 mM Tris, pH 8.0 and 1% Triton ×100. This step was performed inorder to wash out all non-specific proteins, leaving the GST-PAR₁-C-tailcolumn firmly bound to targeted cell lysate proteins. Next, elution ofbound proteins was performed via the addition of gel “sample buffer” andappropriate boiling. The samples were run electrophoretically onSDS-PAGE gels, followed by immunoblotting with the indicated antibodiesand ECL detection. GST-PH-Etk/Bmx. The PH domain in Etk/Bmx was bound toGST column as previously described (Chen R, et al. (2001) Nat Cell Biol.3: 439-444).

Purification of PAR₁ C-tail fragments. PAR₁ C-tail fragments weregenerated using a “thrombin cleavage capture kit” (Novagen, Madison,Wis.; Cat no. 69022-3). The enzyme used for the cleavage wasbiotinylated human thrombin. Briefly, the cleavage was performedaccording to the manufacturer instructions. Biotinylated thrombin wasremoved from the cleavage reaction using streptavidin agarose beads, andthe cleaved peptides (e.g., wt PAR₁-C-tail and Y381A C-tail) wereisolated and loaded on a GST-Etk-PH column. After incubation for 4 h thepurified fragments were applied onto the GST-PH-Etk/Bmx column anddetected following gel separation and western blotting analysis usinganti-PAR₁ antibodies (ATAP, Santa Cruz, Calif.).

Flow Cytometry Analysis. To activate PAR₁, thrombin (1 U/ml was addedfor 5 min. The cells were detached from the plates with 0.5 mM EDTA in0.1 M sodium phosphate at pH 7.4 (Biological Industries), washed andre-suspended in PBS. The cells were analyzed by FACS after incubationfor 60 min at 4° C. with 10 μg/ml anti-PAR₁-wede-PE antibodies.

Western blot and immunoprecipitation analysis: Cells were activated withagonist peptide TFLLRNPNDK (SEQ ID NO: 35) for the indicated periods oftime and solubilized in lysis buffer containing 10 mM Tris-HCl, pH 7.4,150 mM NaCl, 1 mM EDTA, 1% TritonX-100, and protease inhibitors (5 mg/mlaprotinin, 1 mM phenylmethylsulfonylfluoride, and 10 mg/ml leupeptin) at4° C. for 30 min. The cell lysates were subjected to centrifugation at12,000 rpm at 4° C. for 20 min. We used 400 μg of the supernatants withanti-PAR₁ (ATAP, Santa Cruz, Calif. 1 μg), anti-HA (anti-HA sc-7392;Santa Cruz, Calif.), anti-Shc or Etk/Bmx antibodies (10 μg/ml). Afterovernight incubation, Protein A-Sepharose beads (Amersham PharmaciaBiotech, Buckinghamshire, UK) were added to the suspension (50 μl),which was rotated at 4° C. for 1 h. The immunocomplexes were eluted andrun electrophoretically on a 10% SDS-PAGE gel, followed by transfer toan Immobilon-P membrane (Millipore). Membranes were blocked and probedwith 1 μg/ml amounts of the appropriate antibodies as follows: anti-PAR₁thrombin receptor mAb, (ATAP, from Santa Cruz, 1:1000); anti-Shc (BD,1:2000); anti-Bmx (Transduction Laboratories, 1:1000) or anti-PY(Upstate 4G10, 1:2500), suspended in 3% BSA in 10 mM Tris-HCl, pH 7.5,100 mM NaCl, and 0.5% Tween-20. After washes the blots were incubatedwith secondary antibodies conjugated to horseradish-peroxidase.Immunoreactive bands were detected by enhanced chemiluminescence (ECL).Membranes were stripped and incubated with anti-IP antibodies to ensureequal protein load.

Anti-PAR₁ polyclonal antibodies were generated using two regions of theN-terminus portion R/SFFLRN by the synthetic peptidesNH₂-CLLRNPNDKYEPFWED-COOH (SEQ ID NO: 50) and NH₂-KSSPLQKQLFAFISC-COOH(SEQ ID NO: 51).

Antibody array: A custom-made antibody array (hypromatrix) containingthirty antibodies against various proteins was prepared (FIG. 1). Theantibodies were immobilized on a membrane, each at a pre-determinedposition, while retaining their capabilities of recognizing andcapturing antigens as well as antigen-associated proteins. MDA-MB-435cells were activated for 10 min with thrombin (1 U/ml). Untreated oractivated cells were lysed with Triton extraction solution: 15 mM Tris,pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.1 mM DTT, 0.5%Triton X-100, 10 leupeptin and 0.5 mM PMSF. Protein extract wasincubated on pre-blocked membrane for 2 h at room temperature. Theantibody array was washed with TBST and incubated with biotinylated PAR₁antibody (ATAP) for 2 h at room temperature. The antibody array waswashed again with TBST, and the membrane was incubated withHRP-conjugated streptavidin for 1 h. Protein-protein interactions weredetected by ECL and exposure to X-ray film.

Matrigel invasion assay: Blind-well chemotaxis chambers with 13-mmdiameter filters were used for this assay. Polyvinylpyrrolidone-freepolycarbonate filters, 8 mm pore size (Costar Scientific Co., Cambridge,Mass.), were coated with basement membrane Matrigel (25 μg/filter).Briefly, the Matrigel was diluted to the desired final concentrationwith cold distilled water, applied to the filters, and dried under ahood. Cells (2×10⁵) suspended in DMEM containing 0.1% bovine serumalbumin were added to the upper chamber. Conditioned medium of 3T3fibroblasts was applied as a chemo-attractant and placed in the lowercompartment of the Boyden chamber. Cells were incubated for 18 h onfilters at 37° C. in 5% CO₂. At the end of the incubation, cells on theupper surface of the filter were removed by wiping with a cotton swab.The filters were fixed and stained with DifQuick System (Dade BehringInc., Newark, N.J.). Ten fields were chosen from the lower surface ofthe filter and cells within each field were counted. The mean+/−SD ofthe ten fields was calculated for each filter. Each assay was performedin triplicate.

MCF10A morphogenesis assay: MCF10A cells were maintained in DMEM/F12medium with 20% donor horse serum. The cells for spheroid assay(DMEM/F12 supplemented with 2% donor horse serum, 10 μg/ml insulin, 1ng/ml cholera-toxin, 100 lag/ml hydrocortisone, 50 U/ml penicillin and50 μg/ml streptomycin) were resuspended at a concentration of 10⁵ cellsper 4.0 ml. Eight-chambered RS glass slides (Nalgene) were coated with35 μl Matrigel per well and left to solidify for 15 min. The cells weremixed 1:1 with assay medium containing 4% Matrigel and 10 ng/ml EGF, and400 μl were added to each chamber of the Matrigel-coated eight-chamberedslide. Assay medium containing SFLLRNPNDK PAR₁ activation peptide (SEQID NO: 52) and 5 ng/ml EGF was replaced every 4 days. The images weretaken between days 8-12. In the representative experiment shown imageswere taken on day 10. The media and supplements were replaced every 4days and thus, the activating peptide was added fresh to the mediumevery 4 days.

Example 2 The Cytoplasmic Tail of PAR1 is Involved in Tumor PromotionVia Binding of Etk/Bmx

PAR₁-enhanced tumor growth and angiogenesis in vivo is abrogated in thepresence of a truncated PAR₁ form. To investigate the role of PAR₁signaling in breast tumor growth and vascularization in vivo, wt hPar1and deletion constructs [e.g., L369Z, which lacks the entire cytoplasmictail, and Y397Z, which exhibits persistent signaling due to impairedinternalization (Shapiro M J, et al (1996) J. Biol. Chem. 271:32874-32880; Hammes SR, and Coughlin SR (1999) Biochemistry 38:2486-2493)] (FIG. 2A) were over-expressed in MCF7 cells. RT-PCR analysisof the cells transfected with wt hPar1, Y397Z hPar1, truncated hPar1 orempty vector was performed as described above using primers to PAR₁N-terminus, C-terminus, or GAPDH. As can be seen in FIG. 2B thetransfected cells (except for the empty vector) expressed N-terminusPAR1. As expected, only the cells that were transfected with wt hPar1expressed the c-tail portion of PAR1. The functional outcome of MCF7cells over-expressing various hPar1 constructs in vivo was assessed byanalyzing orthotopic mammary fat pad tumor development. MCF7 cellsover-expressing either Y397Z or wt hPar1 constructs (e.g., MCF7/Y397ZhPar1; MCF7/wt hPar1) markedly enhanced tumor growth in vivo followingimplantation into the mammary glands (FIGS. 2C and 2D), whereas MCF7cells over-expressing truncated hPar1 behaved similar to control MCF7cells in vector-injected mice, which developed only very small tumors(FIG. 2C). The tumors obtained with MCF7/wt hPar1 and MCF7/Y397Z hPar1were 5 and 5.8 times larger, respectively, than tumors produced by theMCF7/empty vector-transfected cells. Histological examination (H&Estaining) showed that while both MCF7/wt hPar1 and MCF7/Y397Z hPar1tumors infiltrated into the fat pad tissues of the breast, theMCF7/Y397Z hPar1 tumors further infiltrated the abdominal muscle (FIG.2D). In contrast, tumors produced by empty vector or truncatedhPar1-transfected cells were capsulated, with no obvious cell invasion.In addition, the Y397Z hPar1 and wt hPar1 constructs exhibited intensevascularization and appeared reddish as opposed to the pale appearanceof tumors generated by either empty vector or truncated hPar1.Proliferation levels were evaluated by immunostaining with Ki-67 andwere 3 times higher in Y397Z hPar1 or wt hPar1 tumors (FIG. 3) than inthe small tumors produced by either empty vector or truncatedhPar1-transfected cells (p<0.0001, FIG. 3B). Tumor growth can also beattributed to blood vessel formation (Griffin C T, et al (2001) Science293: 1666-1670; Connolly A J, et al (1996) Nature 381: 516-519). ThehPar1-induced breast tumor vascularization was assessed byimmunostaining with anti-lectin- and anti-CD31 antibodies. BothMCF7/Y397Z and MCF7/wt hPar1 tumors were intensely stained (FIGS. 3A, 3Cand 3D). In contrast, only a few blood vessels were found in the smalltumors of empty vector or truncated hPar1 (FIGS. 3A, 3C and 3D). Thus,both MCF7/wt hPar1 and MCF7/Y397Z hPar1 cells were shown to effectivelyinduce breast tumor growth, proliferation and angiogenesis, while theMCF7/truncated hPar1 and MCF7/empty vector-expressing cells had nosignificant effect. Histological evaluation and scoring was performed asdescribed above.

PAR₁ C-tail binds the She adaptor protein. To identify proteins thatassociate with the PAR₁ C-terminus and participate in the tumorsignaling pathway, the cytoplasmic tail of hPar1 was fused to a GSTprotein and used as “bait” to specifically detect associated proteins.Lysates obtained from a highly metastatic breast carcinoma line (e.g.,MDA-MB-435 cells) were assessed for binding to the GST-PAR₁ C-tailcolumn. After an adequate binding period of the designated cell lysatesto the columns, a washing step was performed. This step was performed inorder to wash out all non-specific proteins, leaving the GST-PAR₁-C-tailcolumn firmly bound to targeted cell lysate proteins. Next,specifically-bound proteins were eluted via the addition of gel “samplebuffer” and detected by Western blot analysis using anti-Shc antibodies.Amino acid sequence analysis of proteins bound to the column repeatedlyindicated the presence of the Shc adapter protein. Indeed, applicationof MDA-MB-435 cell lysates onto a GST PAR₁ C-tail column or a GSTcontrol column showed the three Shc isoforms specifically bound to theGST-PAR₁-C-tail column, but not to the GST control column (FIG. 4A). Shcisoforms refer to a series of proteins (e.g., 66, 52 and 46 kDa) termedShc (Src homology 2/α-collagen-related) (Pelicci G, et al. (1992) Cell70: 93-104: Pronk G J, et al (1993) J Biol. Chem. 268: 5748-5753). cDNAanalyses of the family proteins has demonstrated that the 46- and 52-kDaspecies arise from alternative translation initiation sites within thesame transcript, giving rise to a 59-amino acid terminal truncation ofthe 46-kDa isoform compared to the 52-kDa isoform. In contrast, the66-kDa species most likely arises from an alternatively spliced messagesince there is only one Shc gene and the carboxy terminal antibodiescross react with all three molecular weight species.Co-immunoprecipitation studies using either PAR₁ (FIG. 4B) or Shcantibodies (FIG. 4C) confirmed the PAR₁-Shc association 5 min afterTFLLRNPNDK activation; this association remained high during the 30 minof analysis (FIGS. 4B and C). The Shc protein comprises multiple proteindocking sites, including SH2, phospho-tyrosine binding site (PTB) andcollagen homology domains 1 and 2 (CH1, CH2). When a GST-Shc-SH2 domainpull-down assay was used following loading with PAR₁ activatedMDA-MB-435 cell lysates, PAR₁-specific binding to the Shc-SH2 domain wasobtained. In contrast, when the tandem SH2 domain from an irrelevantprotein was used as a control, no binding of PAR₁ was observed (FIG.4D). Using the NetPhos 2.0 server four candidates: Y₃₈₁, Y₃₈₃, Y₃₉₇,Y₄₂₀ (FIGS. 4E and 4F) were identified as PAR₁ C-tail putative tyrosineresidues capable of serving as possible binding sites for the Shcprotein. Of these, only three were predicted to undergo phosphorylation:Y₃₈₁, Y₃₉₇ and Y₄₂₀ (FIG. 4F). Since, as shown above, Y397Z hPar1 waspotent in signaling (FIGS. 2 and 3), it was postulated that the Shcbinding site(s) in the PAR₁ C-tail is/are located upstream of tyrosine397. Indeed, sequence alignment of PAR₁ C-tail in nine different speciesdemonstrates several highly conserved regions (data not shown), amongwhich are the Y₃₈₁VY₃₈₃ residues. Replacement of the relevant tyrosine(Y) residues upstream to Y397Z hPar1 with alanine (Ala, A) (e.g., Y₃₈₁Aor Y₃₈₃A and the double mutant Y₃₈₁A & Y₃₈₃A) did not prevent therecruitment and physical association between Shc and PAR₁ (see FIG. 9A).

Y₃₈₁A-hPar1 exhibits high metastatic potential. The functionality of theY₃₈₁A hPar1 mutant was further demonstrated in vivo using acolorectal-liver metastasis model (Qiu Y, et al (1998) Proc Natl AcadSci USA 95: 3644-3649), which provides a rapid metastatic model of liverfoci formation. Mouse CT-26 colon carcinoma cells (that do not expressendogenous hPar1) were genetically engineered to over-express wt hPar1,Y₃₈₁A hPar1 or empty vector constructs. These over-expressing cells wereinjected intra-splenically into CB6F1 mice (either PAR₁-activated ornot) to generate liver metastases. Tumor growth kinetics and livermetastatic foci appearance were monitored twice a week by MRI. Both wthPar1 and Y₃₈₁A hPar1 enhanced liver metastatic foci formation, comparedto control CT-26 cells. Furthermore, mice inoculated with cellsexpressing Y₃₈₁A hPar1 showed especially extensive and rapid appearanceof liver metastasis as compared to mice inoculated with cellsover-expressing wt hPar1 (FIG. 5). Representative MRI images (FIG. 5A)of excised livers and histological sections (FIG. 5B), obtained on day16, demonstrated high metastatic potential of both activated Y₃₈₁A hPar1and wt hPar1. An elevated number of metastatic foci were observed withthe wt hPar1 after PAR₁ activation (FIG. 5A), and an even more dramaticincrease was obtained with the activated Y₃₈₁A hPar1 construct (FIGS.5A, C). Quantification of liver metastasis as a function of time isshown in FIG. 5C. These results emphasized that the Y₃₈₁A hPar1 mutatedconstruct is at least as functional as the wt hPar1, and thesubstitution of Y to A did not impair the ability of hPar1 to initiatesignaling and therefore result in metastasis. The results may furthersuggest that Shc does not bind directly to PAR₁ C-tail, sincereplacement of a key tyrosine residue by alanine (Y381A) does not impairPAR₁ function as manifested by metastatic foci formation. It is thuspostulated that whereas Shc is not associated with PAR1 via thetraditional tyrosine-phosphorylated-SH2 complex formation, it probablyinvolves a third mediator connecting with PAR₁. The molecular mechanismof Y₃₈₁A hPar1-enhanced liver metastasis remains to be fully elucidated.

Antibody-array for protein-protein interactions reveals signalingcandidates. To detect the putative mediator(s) linking PAR1 to potentialsignaling proteins, custom-made antibody-array membranes were examined.Aggressive breast carcinoma MDA-MB-435 cells (with high hPar1 levels)were incubated with the antibody-array membranes before and after PAR₁activation (15 minutes). Several activation-dependent proteins whichinteract with PAR₁ were identified, including ICAM, c-Yes, Shc andEtk/Bmx (FIG. 1).

Etk/Bmx-PAR1 interactions were characterized by binding lysatesexhibiting various hPar1 forms to GST-Etk/Bmx. While Y397Z hPar1 and wthPar1 showed specific association with Etk/Bmx, lysates of truncatedhPar1 or JAR cells (lacking PAR₁) exhibited no binding (FIG. 6A). Inorder to substantiate the physical association between PAR₁ C-tail andEtk/Bmx-PH domain the C-tail portion of both wt- and Y381AhPar1-modified tail were proteolytically cleaved and the purifiedfragments were applied onto a GST-PH Etk/Bmx column. Specific bindingwas observed with both the wt hPar1 and Y₃₈₁A hPar1 purified C-tails(FIG. 6B). Next, various modified PAR₁-GST-C tail constructs (e.g., wthPar1, Y₃₈₁A hPar1 and Y₃₈₃A hPar1) were analyzed for binding to eitherwt- or kinase-inactive Etk/Bmx (KQ) cell lysates. A tight associationbetween the PAR₁ C-tail and Etk/Bmx was obtained, independent of whetherwt hPar1 or the Y/A mutant forms of PAR₁ C-tail were examined. This wasdemonstrated for both wt- and KQ-Etk mutant (FIG. 6C).

Differential expression of Etk/Bmx in breast biopsies PAR₁ is known tobe highly expressed in breast carcinoma specimens, but not in normalbreast tissue. Immunohistochemical staining of PAR₁ tissue sectionsconfirms the earlier described RNA riboprobe analysis for hPar1.Invasive carcinoma specimens were selected from infiltrating ductalcarcinoma (IDC) of high nuclear grade and with evidence of vascularinvasion and lymph node metastases. Immunohistological analyses of bothPAR₁ and Etk/Bmx showed little staining in comedo DCIS and ductalcarcinoma in situ, but high levels of staining in IDC and lobularcarcinoma (FIG. 6D).

The combined histological results are shown in Table 1. The results wereassessed and scored as outlined in the Materials & Method section. Themeasurements per slide section were carried out using anatomicalcompartments, using an ocular micrometer (WHIOX2, Olympus, N.J., USA).

The microscope was calibrated with a micrometer slide before eachmeasurement. On examining the sections for selection of fields tumorcells from the most cellular area at the center of the tumor wereselected. Necrotic and inflammatory areas were avoided. Eightmicroscopic fields were screened. Ten cells per each field were selectedand no less than 50 cells/tumor case were assessed. The positive rate ofstaining is expressed as a mean±SD per tumor histological subtype fromselected cases. Specific staining is observed in both PAR₁ and Etk/Bmx,with particularly strong staining seen in IDC and lobular carcinoma. Nostaining is seen in the normal breast tissue.

This staining represents a total of 36 cases as outlined for eachhistological subtype in Table 1, performed three times per category.

TABLE 1 Expression of PAR₁ and Etk/Bmx in breast cancer biopsy specimensPositive cells Histological Cases Mean ± SD +1 +2 +3 subtype (N = 36)PAR₁ Etk/Bmx PAR₁ Etk/Bmx PAR₁ Etk/Bmx PAR₁ Etk/Bmx Normal 12 0.8 ± 0.21.2 ± 0.2   0 1 0 0 0 0 DCIS 8 12.5 ± 3.7  14 ± 3.0  2(25%) 1(12.5%)6(75%) 7(87.5%) — — IDC 9   40 ± 10.5 42 ± 12.3 2(22%) 1(11%)   1(11%) 05(55%) 6(66%) Lobular 7  45 ± 7.3 43 ± 11.6 1(14%) 0 1(14%) 1(14%)  6(85%)   5(71.4%) carcinoma Histological scoring of (N) cases: +1 lessthan 25% positive cells (weak positive); +2 between 25-75% positivecells (moderate); +3 more than 75% of positive cells (strong). Allcontrols were negative (0-5% positive cells). Extent of expressionclassified by score (1-3), number of positive cells/field (x = 8).

These results further suggest a direct correlation between PAR₁ andEtk/Bmx expression in malignant breast cancer progression.

Hierarchy of binding Next, the chain of events mediating the signalingof PAR₁ C-tail-Shc and Etk/Bmx was determined. MCF7 cells that expresslittle or no hPar1 were ectopically forced to over-express hPar1. Whenco-immunoprecipitation with anti-PAR₁ antibodies following PAR₁activation was performed, surprisingly, no Shc was detected in the PAR₁immunocomplex (FIG. 7A; MCF7/hPar1; right panel). Shc association withPAR₁ was fully rescued when MCF7 cells were initially co-transfectedwith Etk/Bmx (FIG. 7A), with abundant assembly of Shc in theimmunocomplex. Thus, Etk/Bmx is a critical component that binds toactivated PAR₁ C-tail and enables subsequent binding of Shc. Shc maybind either to phosphorylated Etk/Bmx, via the SH2 domain, or in anunknown manner to the PAR₁ C-tail, provided that Etk/Bmx is present andis PAR₁-bound. One cannot, however, exclude the possibility that Bmxbinds first to Shc and only then does the complex of Etk/Bmx-Shc bind toPAR-1.

Identification of PAR₁-Etk/Bmx binding region: functional consequences.Peptides (representing various regions in PAR₁ C-tail) were used in acompetition analysis assay for the binding of PAR₁ cell lysates toGST-PH-Etk/Bmx. An 18-amino-acid peptide encompassing residues 375-392of PAR₁ C-tail (termed peptide 4) yielded a dose-dependent inhibitionwithin the range of 1-1000 nM applied peptide (FIG. 7B). Two otherpeptides, representing PAR₁ C-tail 387-400 (FIG. 7C; termed peptide 5)or residues 393-412 did not compete.

Based on the competition assay mutated hPar1 constructs with successivereplacement of seven residues (378-384; CQRYVYS) were prepared. MCF7clones expressing either HA-hPar1-7A or HA-wt hPar1 were prepared. Asshown in FIG. 8 the transfected clones properly expressed HA-PAR1. FIG.9A shows a schematic representation of wt hPar1 and the mutant hPar1-7A.The clones expressing either HA-tagged wt hPar1 or a mutant construct ofHA-hPar1-7A were activated and further analyzed forco-immunoprecipitation (IP) of PAR₁ with Etk/Bmx. The IP was carried outusing anti-HA (sc-7392; Santa Cruz, Calif.). The blots were detected byanti-Bmx (Transduction Laboratories, BD Biosciences, CA) for theidentification of Etk/Bmx-associated PAR₁. As shown in FIG. 9B ectopicexpression of the hPar1-7A mutant construct abrogated binding of Etk/Bmxto PAR₁ C-tail. We thus conclude that the critical region for Etk/Bmxbinding to PAR₁ C-tail resides in the vicinity of CQRYVYS. Levels of theHA-tag (for PAR₁) are shown in the middle panel of FIG. 9B. Similarly,levels of PAR₁ are also shown by application of anti-PAR₁ (ATAP; SantaCruz, Calif.) (lower panel). The right section shows levels of plasmidtransfection efficiencies in the cells, as indicated by HA-PAR₁ andEtk/Bmx analysis by western blots. As seen, only the wt hPar1co-immunoprecipitated with Etk/Bmx (wt hPar1), while the mutantHA-hPar1-7A clone (mutant clone hPar1-7A) failed toco-immunoprecipitate. This takes place under conditions whereby bothconstructs are well expressed, as evidenced by anti-HA-antibodies(western blot; right panel).

MCF7 cells stably expressing HA-wt-hPar1 and Etk/Bmx, or HA-hPar1-7Amutant and Etk/Bmx were examined in a Matrigel invasion assay. Invadingcells were counted and the mean±SD of ten fields per filter wasdetermined. Stable HA wt hPar1 clone or HA-hPar1-7A mutant clone wereco-transfected with Etk/Bmx construct and TFLLRNPNDK-activated. MarkedMatrigel invasion is seen in the wt hPar1 and Etk/Bmx clones (FIG. 9C).Low invasion levels are observed in the presence of hPar1-7A mutant andEtk/Bmx, hPar1 alone, empty vector, vector and Etk/Bmx or PAR₁antagonist. These data are representative of three experiments.

In a parallel experiment, a wound assay for determining the rate of cellmigration, showing the ability of the cells to fill in gaps in anMDA-MB-435 cell monolayer, was performed. MDA-MB-435 cell monolayerexpressing endogenous Etk/Bmx was scratched to introduce an equalgap-area under the following conditions: control untreated cells,TFLLRNPNDK-activated or SiRNA-Etk/Bmx and TFLLRNPNDK-activated. Rapidclosure of the wound was obtained 24 hours under PAR₁ activation ascompared to control untreated cells. In contrast, no migration andclosure of the wound was seen when the endogenous Etk/Bmx level of thecells was knocked down using an siRNA-Etk/Bmx construct (FIG. 9D, toppanel) and they were TFLLRNPNDK-activated. In another set of woundexperiments, attenuated wound closure was seen in the presence of the Vantagonist SCH79797 and SFLLR NPNDK PAR₁ activation for 24 h, similar tothe control untreated cells. These data are representative of fourexperiments. Similar inhibition was obtained in the presence of the PAR₁antagonist, SCH 79797 (FIG. 9D, bottom panel), pointing to the importantrole of both PAR₁ and Etk/Bmx in wound closure/migration ofPAR₁-activated MDA-MB-435 cells.

FIG. 9E shows results of RT-PCR analysis showing the level of Etk/Bmx inMDA-MB-435 cells before and after SiRNA-Etk/Bmx cell infection.

The highly ordered tissue organization of normal epithelia isaggressively disrupted in pathological conditions. This is wellrecapitulated in the MCF10A cell-growth model, mimicking epitheliaapico-basal polarity (Desgrosellier J S, et al. (2009) Nat Med 15:1163-1169). The morphogenesis of MCF10A mammary acini inthree-dimensional (3-D) basement membrane cultures was examined.Normal-appearing intact spheroids are formed in the presence of controlEtk/Bmx-expressing MCF10A cells and activation by SFLLRNPNDK, a PAR₁agonist peptide (FIG. 10A; a, b). In contrast, in the presence ofectopically forced hPar1 (expressing also Etk/Bmx) and following PAR₁activation, an oncogene-like, migratory morphogenesis was obtained whichwas characterized by a complete loss of the cell-cell tight junctioncontacts and the invaded basement membrane architecture (FIG. 10A; c andg). Significantly, when the MCF10A cells (in the presence ofendogenously expressed Etk/Bmx) were infected with the mutantcytoplasmic form of hPar1 (hPar1-7A) and SFLLRNPNDK PAR₁-activated,nearly normal-appearing spheroid morphology was obtained (FIG. 10A; d,f). This outcome highlights the fact that by preventing immobilizationof Etk/Bmx on PAR₁ C-tail, inhibition of invasion and lack ofapico-basal polarity morphogenesis of an oncogene-like phenotype inMCF10A cells are observed.

Analyses of hPAR1 7A mutants in the xenograft animal model In vivoexperiments were performed with hPAR1 7A mutants in the mammary glandxenograft model describe above. Stable MCF-7 clones were generated.These clones expressed: wt hPar1 or hPar1 7A mutant in the presence orabsence of Etk/Bmx. As described above, these clones were furtheranalyzed in vivo in the xenograft animal model. The mice were preimplanted prior to injection with 17-β-estradiol pellets. Next theclones were orthotopically injected to the mice mammary glands.Enormously large tumors were generated in mice injected with cellsexpressing hPar1-Etk/Bmx. The experiment was stopped at an early stage(e.g., 20 days post injection) due to heavy tumor burden. In-contrast,very small tumors, similar to the empty vector injected cells weregenerated in the presence of hPar1-7A mutant, incapable of bindingEtk/Bmx. Thus, as shown above for the MCF7/Y397Z hPar1 cells, it isdemonstrated that when hPar1 is unable to bind Etk/Bmx, very small to notumors are generated (FIG. 11). Tumor weight is shown by the histograms(FIG. 11B).

Immunohistological Evaluation of the Tumor Sections

The tumors generated by the different clones were sectioned andhistologically examined. Cell proliferation levels in the tumor sectionswere evaluated by immunostaining with anti Proliferating Cell NuclearAntigen (PCNA) antibodies. A high nuclear staining level wasdemonstrated in wt hPar1+ Etk/Bmx tumors (p<0.0001, FIG. 12C) ascompared with the small tumors produced by hPar1-7A mutant+ Etk/Bmxshowing low level of staining (FIG. 12D). Apparently, while an extensiveproliferation rate occurs in the wt hPar1 tumor sections, cellproliferation is dramatically attenuated in the hPar1 7A mutant +Etk/Bmxtumors. In-contrast, immunostaining performed by applying anti caspase 3antibodies showed markedly enhanced cytoplasmic staining in the smallappearing tumors generated by the hPar1-7A mutant+Etk/Bmx (p<0.0003,FIG. 12D). Caspases are well known for their central part in apoptosis.Without wishing to be bound by theory, it seems that apoptosis may beactively on-going in the small appearing tumors generated by hPar1 7Amutant. In-contrast, no apoptosis was observed in the wt hPar1 generatedtumors. When the levels of β-catenin were evaluated, distinctly highnuclear localization of β-catenin was observed in the wt hPar1+ Etk/Bmxtumors. In-contrast, weak cytoplasmic staining was seen in hPar1-7Amutant+Etk/Bmx small tumors (FIG. 12D). These data reaffirm that wthPar1 in the presence of Etk/Bmx generated large tumors while hPar1-7Amutant+Etk/Bmx were incapable of producing growing tumors. Rather, themutant hPar1-7A+Etk/Bmx showed a reduced proliferation rate, enhancedapoptotic levels and low levels of β-catenin (an indicator ofoncogenicity) localized in the cytoplasmic fraction of the cells. Theidentification of a PAR₁ C-tail binding domain provides a platform fornew therapeutic vehicles in the treatment of cancer, and in particularbreast cancer.

Example 3 The PAR₂ C-Tail Minimal Binding Site for Etk/Bmx

HEK-293T cells were transiently transfected with both hPar2 andT7-tagged Etk/Bmx. Immunoprecipitation analyses were performed beforeand after PAR₂ activation with SLIGKV (a peptide known to activatePAR₂). The results showed a firm association between PAR₂ and Etk/Bmx,occurring between 10-20 min of activation which declined immediatelythereafter (FIG. 13 a). Similar outcome was obtained in MCF7 cellstransfected to express both hPar2 as well as Etk/Bmx (FIG. 13 b). Thesedata clearly indicate that PAR₂ behaves in a similar manner as PAR₁following activation and associates with Etk/Bmx.

The physical association between Etk/Bmx and PAR₂ was confirmed in assayexamining the binding between Etk/Bmx expressing cell lysates andGST-PAR₂ C-tails. A truncated form of hPar2 showed no physicalinteraction between PAR₂ and Etk/Bmx as compared with wt hPar2 (FIG.14A). Various PAR₂ deletion constructs were used to identify the minimalbinding domain within PAR₂ C-tail as shown in FIG. 14B. Lanes: A.control, B. GST-PAR2c-tail wt; C. GST-PAR₂C-tail K378Z; D.GST-PAR₂C-tail K3356Z. Apparently, the smallest c-tail analyzed, K365Z,exhibits effective association. The minimal signal binding region in thePAR₂ C-tail was found to be: NH2-SHDFRDHA-COOH.

Example 4 Inhibition of PAR₂ Signaling In Vivo

The physiological significance of a prime signaling partner thatinteracts with PAR₂ C-tail is assessed in vivo, in an orthotopic mammaryfat pad model for tumor development. As shown above in example 2, stableMCF7 clones expressing various constructs of hPar2 are generated andinjected orthotopically into mice, mammary glands. These clones includewt hPar2, truncated hPar2 (devoid of the cytoplasmic tail) and thevarious deleted hPar2-C-tail constructs (e.g., K390Z hPar2; K378Z hPar2,K368Z hPar2 and K356Z hPar2). In parallel, a silenced siRNA-hPar2lentiviral construct is evaluated for the ability to halt tumor growth,in vivo. For the cloning of siRNA-hPar2 into a lentiviral vector thefollowing primers are used

P-TG GGAAGAAGCCTTATTGGTA TTCAAGAGATACCAATAA GGC TTCTTCCCTTTTTTC (SEQ IDNO: 53)

5′-P-TCGAGAAAAAAG GGAA GAAGCC TTATTGGTATC TCTTGAATACCAATA AGGCTTCTTCCCA(SEQ ID NO: 54). Stable clones expressing both wt hPar2 and the mutatedhPar2 C-tail (incapable of binding a prime signaling partner; e.g.Etk/Bmx) are generated. These clones are analyzed in vivo to evaluatethe physiological significance for preventing a prime signaling partnerassociation with PAR₂.

Example 5 Selective Association of Additional PH-Domain ContainingSignal Proteins with PAR₁ and PAR₂

The association between Etk/Bmx and PAR₁-C-tail occurs via Etk/Bmx'sPH-domain. Next, the association of additional PH-domain containingproteins, e.g. Akt and Vav3 with PAR₁ or PAR₂ was examined. FIG. 15shows the sequence alignment of the PH domain of the proteins Etk/Bmx,vav3 and Akt.

First, the endogenous level of Akt and Etk/Bmx was analyzed in varioustumor cell lines (i.e. HEK-293T, CL1, HCT116, MDA-MB-231, andMDA-MB-435). While Akt is abundantly expressed in all lines tested,Etk/Bmx appeared endogenously only in CL1, a prostate cancer cell line(FIG. 16A). Next, binding immobilization of cell lysates followingapplication on GST beads of either PAR₁-C-tail or PAR₂-C-tail wasemployed. While no binding was observed on a GST column alone, specificbinding was seen in HEK-293T cells that were ectopically transfected toexpress Etk/Bmx. This binding was found in both GST-PAR₁-C-tail and alsoin GST-PAR₂-C-tail. However, in HCT-116 cells lacking Etk/Bmx no suchbinding was seen but rather both PAR₁ and PAR₂ C-tails effectivelyassociated with Akt (FIG. 16B). Without wishing to be bound by theory,these observations suggest that both PAR₁ and PAR₂ C-tails bindprimarily to Etk/Bmx, but in instances where Etk/Bmx is absent, Akt ispotently associated with both PARs C-tails.

Next, a panel of cell-lines was transfected with HA-tag-hPar1. Celllysates were prepared and applied onto GST-PH-Akt or GST-PH-Vav₃.HEK-293T cells were used as naïve parental cells (e.g., lacking Etk/Bmx)as also following enforced expression of Etk/Bmx. As shown in FIG. 17Acells that do not express endogenously Etk/Bmx, are capable ofeffectively associating with PH-Akt (e.g., HCT-116 and HEK-293T cells).In-contrast, cells that express Etk/Bmx either endogenously (e.g., CL1)or following transfection (e.g., HEK-293T cells) fail to physicallyassociate with PAR₁. Similarly the same pattern of association wasobserved when the cell lysates were applied onto GST-PH-Vav₃ (FIG. 17A).In order to substantiate the observation that Etk/Bmx preferentiallyassociates with PAR₁-C-tail, increasing concentrations of Etk/Bmx weretransfected into HEK-293T cells. Indeed, while naïve HEK-293T cellsdisplayed potent binding with the PH-domain of either. Akt or Vav₃, thepresence of Etk/Bmx abrogated these associations, at the lowestconcentration used for transfection (e.g., 0.5 μg) (FIG. 17C). Thisoutcome supports the hypothesis that Etk/Bmx associates preferentiallywith PAR₁-C-tail, however that it is capable of associating with otherPH-domain containing signal proteins.

The PH-Domain Binding Region in PAR₁-C-Tail

The following example demonstrates that the association of PAR₁-C-tailwith PH-domain containing proteins, e.g. PH-domain-Akt or PH-domain-Vav₃occurs via the same region that is associated with Etk/Bmx. Namely,without wishing to be bound by theory the PAR₁-C-tail appears to be ananchor region for association with PH-domain containing proteins. Forthis purpose, the association between Akt (which is ubiquitouslyexpressed in the cells) and PAR₁ was analyzed, following activation. Asshown in FIG. 18, Co-immunoprecipitation between Akt and PAR₁ wasobtained following 2 and 5 minutes activation, and declined immediatelythereafter. Nearly normal epithelial HU cells were next transfected witheither wt hPar1 or with the mutant hPar1-7A, incapable of bindingEtk/Bmx. Immunoprecpitation analyses showed that while potentassociation between Akt and PAR₁ was observed in the presence of wthPar1, no association was seen when the mutant hPar1 7A was present(FIGS. 18 A and B). Likewise, when cell lysates expressing either wthPar1 or the mutant hPar1 7A, were applied on GST-PH-Vav₃ an essentiallysimilar pattern of association occurred. While the wt form firmly bindsto PAR₁-C-tail, it completely failed to bind in the presence of themutant PAR₁-7A (FIG. 18C).

The PH-Domain Binding Region in PAR₂-C-Tail

Cells that do not express endogenous Etk/Bmx effectively associated witheither PH-Akt or PH-Vav₃ as demonstrated upon application on GSTimmobilized PH-domain columns. HEK-293 cells effectively bind PH-Akt orPH-Vav₃. In-contrast, when these cells were transfected with increasedconcentrations of Etk/Bmx (e.g., 0.5-2 μg) they completely failed toassociate with either of the PH-domain proteins analyzed (e.g., Akt andVav₃). These data strongly indicate that activation of PAR₂ behaves in asimilar manner as activated PAR₁ and primarily associates with thePH-domain of Etk/Bmx (FIG. 19). Once Etk/Bmx is absent, other signalcontaining PH-domain/s can associate with PAR₂-C-tail as portrayed viabinding to both Akt and Vav₃.

In order to determine the minimal binding region within PAR₂-C-tailsequence, deleted hPar2-C-tail constructs were prepared. HEK-293T cellswere transfected to overexpress either wt hPar2 or the shortest hPar2deleted construct (e.g., hPar2 K356Z) or mutants inserted into theshortest Etk/Bmx, Akt or Vav₃. Slightly weaker binding is observed inthe presence of the shortest region K356A. When mutants were applied tothis region (e.g., K352A, K349A) both mutants failed to bind eitherEtk/Bmx (FIG. 20A) or Akt. While the mutant K352A still showed weakbinding to Vav₃-PH-domain, the mutant K349A exhibited completeabrogation of this binding (FIG. 20B). Immunoprecipitation analysesbefore and after PAR₂ SLIGKV activation showed association (as evidencedby co-immunoprecipitation) between PAR₂ and Akt at 2 and 10 minutesfollowing activation (FIG. 20D). Indeed, when the mutant K352A waspresent it abrogated the association between PAR₂ and Akt (FIG. 20E).Altogether, these observations collectively show that the bindingassociation to the different PH proteins is largely mediated through thesame region within PAR₂-C-tail.

Example 6 PAR₁ Peptide Penetration into MCF7 Cells

A PAR₁ peptide corresponding to the region which associates with thePH-domain (i.e. FITC-SSECQRYVYSIL-COOH (SEQ ID NO: 3) was synthesizedand tagged with GFP (green fluorescence protein). MCF7 cells werepreincubated for various time periods with the FITC-labeled peptide(e.g., 2 h, 4 h, 8 h and 16 h). After 2 h, some of the cells showeduptake of GFP. Maximal uptake was visualized after 8 h and 16 h. Thelevels of the penetration of the GFP-PH-domain PAR₁ peptide into MCF7cells are shown in FIG. 21. As can be seen in FIGS. 21B and C a largenumber of GFP containing cells can be observed, indicating that thepeptide is capable of penetrating into the cells. Next, a competitionexperiment was performed. Stable clones of MCF7 cells expressing eitherHA-PAR₁ T7-tagged Etk/Bmx, and incubated in the presence or absence ofthe PAR₁ PH-domain peptide (i.e. SSECQRYVYSIL). The cells were activatedeither by thrombin or using the PAR₁ activating peptide TFLLRN, and celllyzates were prepared at various time points after activation.Immuno-precipitation analyses were performed with anti HA antibodies(thereby immunopreciptating HA-FARO, followed by Western blot analysisusing anti T7 antibodies (detecting T7-tagged Etk/Bmx). As demonstratedin FIG. 22 the association between Etk/Bmx and PAR₁-C-tail can be seenfollowing 20 minutes activation without the peptide. No binding isobserved in the presence of the peptide under similar conditions. Thusthe peptide is capable of preventing effectively the PAR1 signallingcascade.

1.-28. (canceled)
 29. An isolated PAR1 cytoplasmic tail (c-tail) peptideselected from the group consisting of: a) an isolated PAR1 c-tailpeptide capable of interfering in the binding reaction between PAR1 anda PH-domain containing protein; b) an isolated PAR1 c-tail peptidecomprising SEQ ID NO: 1; c) an isolated PAR1 c-tail peptide consistingof the sequence SSECQRYVYSIL (SEQ ID NO: 3); d) an isolated PAR1 c-tailpeptide consisting of the sequence SSECQRYVYSILCCK (SEQ ID NO: 4); ande) any fragment or modification of the peptides of (a) (b) (c) or (d).30. An isolated PAR1 c-tail peptide according to claim 29 wherein saidPH-domain containing protein is selected from the group consisting ofEtk/Bmx, Akt and vav3.
 31. An isolated PAR1 c-tail peptide according toclaim 30 wherein said PH-domain containing protein is Etk/Bmx.
 32. Anisolated PAR2 c-tail peptide selected from the group consisting of: a)an isolated PAR2 c-tail peptide capable of interfering in the bindingreaction between PAR2 and a PH-domain containing protein; b) an isolatedPAR2 c-tail peptide comprising SEQ ID NO: 2; and c) any fragment ormodification of the peptides of (a) or (b).
 33. A method of inhibitingPAR1 mediated signal transduction comprising administering an agentcapable of selectively inhibiting the binding of PAR1 and a PH-domaincontaining protein.
 34. A method according to claim 33 wherein saidagent is a peptide.
 35. A method of inhibiting PAR2 mediated signaltransduction comprising administering an agent capable of selectivelyinhibiting the binding of PAR2 and a PH-domain containing protein.
 36. Amethod of treating a disease comprising administering a therapeuticallyeffective amount of an agent capable of selectively inhibiting thebinding of PAR1 and a PH-domain containing protein, or an agent capableof selectively inhibiting the binding of PAR2 and a PH-domain containingprotein, or a pharmaceutical composition comprising said agent to apatient in need thereof.
 37. A method according to claim 36 wherein saiddisease is cancer.
 38. A method according to claim 36 wherein said agentis a peptide.
 39. A method according to claim 38, wherein the peptide isselected from the group consisting of: a) An isolated PAR1 c-tailpeptide comprising the amino acid sequence CQRYVYS (SEQ ID NO: 1); b) anisolated PAR1 c-tail peptide consisting of the sequence SSECQRYVYSIL(SEQ ID NO: 3); c) An isolated PAR1 c-tail peptide consisting of theamino acid sequence SSECQRYVYSILCCK (SEQ ID NO: 4); and d) any fragmentor modification of the peptides of (a) (b) or (c).
 40. A methodaccording to claim 38, wherein the peptide is selected from the groupconsisting of: a) An isolated PAR2 c-tail peptide comprising the aminoacid sequence SHDFRDHA (SEQ ID NO: 2); and b) any fragment ormodification of the peptide of (a).
 41. A method according to claim 36further comprising administering an additional therapeutic agent.
 42. Apharmaceutical composition comprising at least one peptide according toclaim 29 and/or at least one peptide according to claim 32 together witha pharmaceutically acceptable carrier or diluent.
 43. A pharmaceuticalcomposition according to claim 42 further comprising an additionaltherapeutic agent.
 44. A pharmaceutical composition comprising: at leastone isolated PAR1 cytoplasmic tail (c-tail) peptide selected from thegroup consisting of: a) an isolated PAR1 c-tail peptide capable ofinterfering in the binding reaction between PAR1 and a PH-domaincontaining protein; b) an isolated PAR1 c-tail peptide comprising SEQ IDNO: 1; c) an isolated PAR1 c-tail peptide consisting of the sequenceSSECQRYVYSIL (SEQ ID NO: 3); d) an isolated PAR1 c-tail peptideconsisting of the sequence SSECQRYVYSILCCK (SEQ ID NO: 4); and e) anyfragment or modification of the peptides of (a) (b) (c) or (d), at leastone isolated PAR2 c-tail peptide selected from the group consisting of:f) an isolated PAR2 c-tail peptide capable of interfering in the bindingreaction between PAR2 and a PH-domain containing protein; g) an isolatedPAR2 c-tail peptide comprising SEQ ID NO: 2; and h) any fragment ormodification of the peptides of (f) or (g), together with apharmaceutically acceptable carrier or diluent.